Maytansinoid-based drug delivery systems

ABSTRACT

The present subject matter provides for albumin-binding prodrugs, maytansinoid-based compounds, and uses thereof.

This application claims priority to U.S. Provisional Application62/593,184 filed Nov. 30, 2017, the disclosure of which is incorporatedby reference herein in its entirety.

BACKGROUND

Many drugs, particularly cancer therapeutics, have a narrow therapeuticwindow, wherein their side effects limit their beneficial effects.Systemic administration of such drugs often results in a limitedtherapeutic effect because the dose required to elicit a more robusteffect results in unacceptable side effects to the patient. This isparticularly critical in the case of those drugs, which possess a highcytotoxic potential, such as cytostatic agents, virostatic agents orimmunosuppressive agents. This is even more critical in the case ofcertain cytotoxic agents that inhibit tumor cell growth in the picomolarrange. These agents are generally too toxic for being used aschemotherapeutics. For example, the tubulin-binding maytansine is highlyeffective in inhibiting tumor cell growth but had failed in variousclinical trials due to an unacceptable toxicity profile.

Numerous research endeavors have looked into delivering a particulardrug at a particular site of action. Often, this approach results in ahigher concentration of the drug at the site of action than would beachieved by systemic administration, while limiting the side effects.

Drug delivery in oncology is of particular interest owing to the narrowtherapeutic window of agents used in such indication. Numerous researchefforts have concentrated on conjugating anticancer drugs with a widespectrum of low- and high-molecular-weight carriers including sugars,growth factors, vitamins, peptides, antibodies, polysaccharides,lectins, serum proteins, and synthetic polymers. In most of these drugdelivery systems, the drug is bound to the carrier through a spacer thatincorporates a pre-determined breaking point that allows the bound drugto be released at the cellular target site (Kratz et al., ChemMedChem,3:20-53 (2008)).

Conjugates are known in which cytostatic agents are bound to serumproteins, predominantly to specific carrier molecules such as humanserum albumin and human serum transferrin, and then administered. Inother instances, conjugates comprising a therapeutically effectivesubstance, a spacer molecule and a protein-binding molecule, bindcovalently to circulating serum albumin upon administration, whichresults in the transport of the therapeutically effective substance tothe target site where it is released (U.S. Pat. No. 7,387,771). In yetother instances, antibody drug conjugates (ADC) can transport the drugto the target site for local release (Kratz et al., ChemMedChem, 3:20-53(2008); Panowski et al., mAbs, 6, 34-45 (2014); Chari et al., AngewandteChem. Int. Ed., 53, 3796-3827 (2014)).

However, when designing drug delivery systems, the appropriate balanceshould be struck between preserving the targeting properties of the drugcarrier while enabling a controlled release of the drug. The drugdelivery system should have sufficient stability in the bloodstream, andyet allow effective release of the drug at the tumor site by enzymaticcleavage, reduction or, in a pH-dependent manner (Kratz et al.,ChemMedChem, 3:20-53 (2008)). For highly potent cytotoxic agents fromthe class of maytansinoids (derived from maytansine), only drug deliverysystems wherein the maytansinoid-based active species is releasednon-specifically or reductively were reported. Among these only thoseusing a monoclonal antibody as the carrier molecule have entered aclinical stage of development and merely one antibody-maytansinoidconjugate, namely T-DM1 (Kadcyla®) has gained market approval againstcertain subtypes of breast cancer. Therefore, there is still a need forefficient and less complex drug delivery and release systems thatrelease highly potent cytotoxic maytansinoid-based agents in aneffective manner.

SUMMARY

The present disclosure provides a compound having the structure ofFormula (I):

-   -   or a pharmaceutically acceptable salt, hydrate, solvate, or        isomer thereof, wherein:    -   R¹ is selected from —H and C₁-C₄ alkyl;    -   Spacer is selected from:

-   -   V is absent or selected from —CH₂—, —O— and —NR³—, wherein R³ is        —H or C₁-C₄ alkyl;    -   each R² is independently selected from —H, halogen (e.g., —F,        —Cl, —Br or —I), and C₁-C₄ alkyl or two R²s taken together form        a C₃-C₆, cycloalkyl;    -   n is 0-3;    -   X is absent or selected from —CH₂—, —O—, —S—, —Se—, and —NR⁴—,        wherein R⁴ is —H or C₁-C₄ alkyl;    -   Y is selected from ═CH— and ═N—;    -   Z¹, Z², Z³ and Z⁴ are each independently selected from —H,        halogen (e.g., —F, —Cl, —Br, or —I), —CF₃, —OCH₃, —CN, —NO₂,        C₁-C₄ alkyl and C₂-C₄ alkoxy;    -   AA is an amino acid selected from glycine, D or L proline,        sarcosine, N-ethyl-glycine, D or L alanine, D or L        N-methylalanine, β-alanine, N-methyl-β-alanine,        α-aminoisobutyric acid, and N-methyl-α-aminoisobutyric acid;    -   R′ is selected from O and

-   -   Y′ is absent or selected from an optionally substituted C₁-C₆        alkyl, —NH—C(O)—, and —C(O)—NH—; or Y′ is selected from the        group consisting of:

wherein n=0-6;

-   -   R^(1′) is absent or selected from the group consisting of:

-   -   wherein M¹ is a pharmaceutically acceptable counter ion (e.g.,        H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, NR₄ ⁺, and NHR₃ ⁺; wherein R is H or        C₁-C₄ alkyl);    -   R^(2′) is optionally substituted C₁-C₁₈ alkyl wherein optionally        up to six carbon atoms in said C₁-C₁₈ alkyl are each        independently replaced with —OCH₂CH₂—;    -   Z^(1′), Z^(2′), Z^(3′) and Z^(4′) are each independently        selected from —H, halogen (e.g., —F, —Cl, —Br or —I) —CF₃,        —OCH₃, —CN, —NO₂, —SO₃M², and C₁-C₄ alkyl wherein M² is a        pharmaceutically acceptable counter ion (e.g., H⁺, Na⁺, K⁺,        Ca²⁺, Mg²⁺, NR₄ ⁺, and NHR₃ ⁺; wherein R is H or C₁-C₄ alkyl);    -   TBG is a thiol-binding group selected from an optionally        substituted maleimide group, an optionally substituted        haloacetamide group, an optionally substituted haloacetate        group, an optionally substituted pyridylthio group, an        optionally substituted isothiocyanate group, an optionally        substituted vinylcarbonyl group, an optionally substituted        aziridine group, an optionally substituted disulfide group, an        optionally substituted acetylene group, and an optionally        substituted N-hydroxysuccininide ester group;    -   wherein said TBG is optionally bound to a thiol-bearing        macromolecular carrier or thiol-bearing tumor-specific carrier.

In some embodiments, the disclosure provides a compound having thestructure of Formula (I):

-   -   or a pharmaceutically acceptable salt, hydrate, solvate, or        isomer thereof, wherein:    -   R¹ is selected from —H and C₁-C₄ alkyl;    -   Spacer is selected from:

V is absent or selected from —CH₂—, —O— and —NR³—, wherein R³ is —H orC₁-C₄ alkyl;

-   -   each R² is independently selected from —H, halogen (e.g., —F,        —Cl, —Br or —I), and C₁-C₄ alkyl or two R²s taken together form        a C₃-C₆, cycloalkyl;    -   n is 0-3;    -   X is absent or selected from —CH₂—, —O—, —S—, —Se—, and —NR⁴—,        wherein R⁴ is —H or C₁-C₄ alkyl;    -   Y is selected from ═CH— and ═N—;    -   Z¹, Z², Z³ and Z⁴ are each independently selected from —H,        halogen (e.g., —F, —Cl, —Br, or —I), —CF₃, —OCH₃, —CN, —NO₂,        C₁-C₄ alkyl and C₂-C₄ alkoxy;    -   AA is an amino acid selected from glycine, D or L proline,        sarcosine, D or L alanine, D or L N-methylalanine, β-alanine,        N-methyl-β-alanine, α-aminoisobutyric acid, and        N-methyl-α-aminoisobutyric acid;    -   R′ is selected from O and

-   -   Y′ is absent or selected from an optionally substituted C₁-C₆        alkyl, —NH—C(O)—, and —C(O)—NH—; or Y′ is selected from the        group consisting of:

wherein n=0-6;

-   -   R^(1′) is absent or selected from the group consisting of:

-   -   wherein M¹ is a pharmaceutically acceptable counter ion (e.g.,        H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, NR₄ ⁺, and NHR₃ ⁺; wherein R is H or        C₁-C₄ alkyl);    -   R^(2′) is optionally substituted C₁-C₁₈ alkyl wherein optionally        up to six carbon atoms in said C₁-C₁₈ alkyl are each        independently replaced with —OCH₂CH₂—;    -   Z^(1′), Z^(2′), Z^(3′) and Z^(4′) are each independently        selected from —H, halogen (e.g., —F, —Cl, —Br or —I) —CF₃,        —OCH₃, —CN, —NO₂, —SO₃M², and C₁-C₄ alkyl wherein M² is a        pharmaceutically acceptable counter ion (e.g., H⁺, Na⁺, K⁺,        Ca²⁺, Mg²⁺, NR₄ ⁺, and NHR₃ ⁺; wherein R is H or C₁-C₄ alkyl);    -   TBG is a thiol-binding group selected from an optionally        substituted maleimide group, an optionally substituted        haloacetamide group, an optionally substituted haloacetate        group, an optionally substituted pyridylthio group, an        optionally substituted isothiocyanate group, an optionally        substituted vinylcarbonyl group, an optionally substituted        aziridine group, an optionally substituted disulfide group, an        optionally substituted acetylene group, and an optionally        substituted N-hydroxysuccininide ester group;    -   wherein said TBG is optionally bound to a thiol-bearing        macromolecular carrier or thiol-bearing tumor-specific carrier.

In some embodiments, the compound has a structure of Formula (II):

-   -   or a pharmaceutically acceptable salt, hydrate, solvate, or        isomer thereof,    -   wherein:    -   each R² is independently selected from —H, and C₁-C₄ alkyl or        two R²s taken together form a C₃-C₆, cycloalkyl;    -   X is absent or selected from —CH₂—, —O—, —S— and —NR³—, wherein        R³ is —H or C₁-C₄ alkyl;    -   Z¹, Z², Z³ and Z⁴ are each independently selected from —H,        halogen (e.g., —F, —Cl, —Br or —I), —CF₃, —OCH₃, —NO₂ and —CH₃.

In some embodiments, the compound has a structure of Formula (III):

-   -   or a pharmaceutically acceptable salt, hydrate, solvate, or        isomer thereof,    -   wherein:    -   each R² is independently selected from —H, and C₁-C₄ alkyl or        two R²s are taken together form a C₃-C₆, cycloalkyl;    -   X is absent or selected from —CH₂—, —O—, —S— and —NR³—, wherein        R³ is —H or C₁-C₄ alkyl;    -   Z¹, Z², Z³ and Z⁴ are each independently selected from —H,        halogen (e.g., —F, —Cl, —Br or —I), —CF₃, —OCH₃, —NO₂ and —CH₃.

In some embodiments, the compound has a structure of Formula (IV):

-   -   or a pharmaceutically acceptable salt, hydrate, solvate, or        isomer thereof,    -   wherein:    -   X is absent or selected from —CH₂— and —NH—;    -   Y is ═CH— or ═N—;    -   Z¹, Z², Z³ and Z³ are each independently selected from —H,        halogen (e.g., —F, —Cl, —Br or —I), —CF₃, —OCH₃, —NO₂ and —CH₃;    -   AA is an amino acid selected from glycine, D or L proline,        sarcosine, N-ethyl-glycine, D or L alanine, D or L        N-methylalanine, β-alanine, N-methyl-β-alanine,        α-aminoisobutyric acid, and N-methyl-α-aminoisobutyric acid.

In some embodiments, the compound has a structure of Formula (IV):

-   -   or a pharmaceutically acceptable salt, hydrate, solvate, or        isomer thereof,    -   wherein:    -   X is absent or selected from —CH₂— and —NH—;    -   Y is ═CH— or ═N—;    -   Z¹, Z², Z³ and Z³ are each independently selected from —H,        halogen (e.g., —F, —Cl, —Br or —I), —CF₃, —OCH₃, —NO₂ and —CH₃;    -   AA is an amino acid selected from glycine, D or L proline,        sarcosine, D or L alanine, D or L N-methylalanine, β-alanine,        N-methyl-β-alanine, α-aminoisobutyric acid, and        N-methyl-α-aminoisobutyric acid.

In some embodiments, R¹ is —H. In some embodiments, at least one of Z¹,Z², Z³ and Z⁴ is not H. In some embodiments, at least one of Z, Z², Z³and Z⁴ is —F or —NO₂. In some embodiments, n is 0 and X is absent. Insome embodiments, n is 0 and X is —CH₂—. In some embodiments, n is 0 andX is —O—, NHMe, or —S—. In some embodiments, the compound is selectedfrom:

or a pharmaceutically acceptable salt, hydrate, solvate, or isomerthereof.

In some embodiments, the pharmaceutically acceptable counter ion isselected from H⁺, Na⁺, K⁻, Ca²⁺, Mg²⁺, NR₄ ⁺, and NHR₃ ⁺; wherein R is Hor C₁-C₄ alkyl.

In some embodiments, R′ is 0. In some embodiments, R′ is:

In some embodiments, the compound is not bound to a thiol-bearingmacromolecular carrier or thiol-bearing tumor-specific carrier. In someembodiments, the compound is bound to a thiol-bearing macromolecularcarrier or thiol-bearing tumor-specific carrier. In some embodiments,the thiol-bearing macromolecular carrier or thiol-bearing tumor-specificcarrier is selected from endogenous albumin, exogenous albumin, anantibody, an antibody fragment, a peptide, a natural or syntheticpolymer, a liposome and a nanoparticle. In some embodiments, TBG is anoptionally substituted maleimide group. In some embodiments, Z^(1′) isselected from —NO₂ or —SO₃M²;

and Y′ is selected from —NHC(O)— or

In some embodiments, R^(1′) is

In some embodiments, R′ is:

or a pharmaceutically acceptable salt, hydrate, solvate, or isomerthereof,wherein R² is selected from optionally substituted C₁-C₁₈ alkyl whereinoptionally up to six carbon atoms in said C₁-C₁₈ alkyl are eachindependently replaced with —OCH₂CH₂—.

In some embodiments, R′ is:

or a pharmaceutically acceptable salt, hydrate, solvate, or isomerthereof.

In some embodiments, the compound is selected from:

or a pharmaceutically acceptable salt, hydrate, solvate, or isomerthereof.

In some embodiments, R′ is:

or a pharmaceutically acceptable salt, hydrate, solvate, or isomerthereof, wherein M¹ is a pharmaceutically acceptable counter ion.

In some embodiments, the compound is selected from:

or a pharmaceutically acceptable salt, hydrate, solvate, or isomerthereof.

In some embodiments, the compound of any of claims 14-20, wherein R′ is:

or a pharmaceutically acceptable salt, hydrate, solvate, or isomerthereof;wherein M¹ is a pharmaceutically acceptable counter ion.

In some embodiments, the compound of claim 26, wherein the compound is:

Other embodiments include a pharmaceutical composition comprising acompound as disclosed herein, and a pharmaceutically acceptable carrier.

Other embodiments include a method for treating a disease or conditionselected from a cancer, a virus disease, autoimmune disease, acute orchronic inflammatory disease, and a disease caused by bacteria, fungi,or other micro-organisms, comprising administering to a patient in needthereof a therapeutically effective amount of a compound or apharmaceutical composition as disclosed herein. In some embodiments, thedisease is cancer, e.g., a cancer is selected from adenocarcinoma, uvealmelanoma, acute leukemia, acoustic neuroma, ampullary carcinoma, analcarcinoma, astrocytoma, basalioma, pancreatic cancer, connective tissuetumor, bladder cancer, bronchial carcinoma, non-small cell bronchialcarcinoma, breast cancer, Burkitt's lymphoma, corpus carcinoma, CUPsyndrome, colon cancer, cancer of the small intestine, ovarian cancer,endometrial carcinoma, gallbladder cancer, gallbladder carcinoma,uterine cancer, cervical cancer, neck, nose and ear tumors,hematological neoplasia, hairy cell leukemia, urethral cancer, skincancer, gliomas, testicular cancer, Kaposi's sarcoma, laryngeal cancer,bone cancer, colorectal carcinoma, head/neck tumors, colon carcinoma,craniopharyngeoma, liver cancer, leukemia, lung cancer, non-small celllung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, stomach cancer,colon cancer, medulloblastoma, melanoma, meningioma, kidney cancer,renal cell carcinomas, oligodendroglioma, esophageal carcinoma,osteolytic carcinomas and osteoplastic carcinomas, osteosarcoma, ovariancarcinoma, pancreatic carcinoma, penile cancer, prostate cancer, tonguecancer, ovary carcinoma, and lymph gland cancer.

Other embodiments include a method of reducing cytotoxicity of acompound comprising administering a compound or a pharmaceuticalcomposition as disclosed herein to a patient in need thereof, whereinthe administration results in a reduction in cytotoxicity when comparedto an equivalent dose of the unmodified active agent.

Other embodiments include a method of increasing the concentration of ametabolite of a compound in a tumor, comprising administering thecompound or a pharmaceutical composition as disclosed herein to apatient in need thereof, wherein the increase is compared to anequivalent dose of the unmodified active agent.

Other embodiments include a compound as disclosed hereinfor use as amedicament.

Other embodiments include a compound as disclosed hereinfor use intreating a disease or condition selected from the group consisting of acancer, a virus disease, autoimmune disease, acute or chronicinflammatory disease, and a disease caused by bacteria, fungi, or othermicro-organisms.

Other embodiments include a use of a compound or a composition asdisclosed herein in the preparation of a medicament for the treatment ofa disease or condition selected from a cancer, a virus disease,autoimmune disease, acute or chronic inflammatory disease, and a diseasecaused by bacteria, fungi, or other micro-organisms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the stability of different linkers with 4 in CD1 murineplasma.

FIG. 2 shows the heat map of geometric mean IC₅₀ values in a panel ofdifferent cell lines.

FIG. 3 shows tumor growth curves of the control group, maytansine group,and the groups treated with compounds 30, 42, 31 and 35 in the RXF631renal cell tumor model.

FIG. 4 shows curves of body weight change in the control group,maytansine group, and the groups treated with compounds 30, 42, 31 and35 in the RXF631 renal cell tumor model.

FIG. 5 shows tumor growth curves of the control group, maytansine group,and the groups treated with compounds 32, 30, and 31 in the LXFE 937squamous cell lung carcinoma model.

FIG. 6 shows curves of body weight change in the control group,maytansine group, and the groups treated with compounds 32, 30, and 31in the LXFE 937 squamous cell lung carcinoma model.

FIG. 7 shows tumor growth curves of the control group, maytansine group,and the groups treated with compounds 30 and 31 in the LXFE 937 squamouscell lung carcinoma model.

FIG. 8 shows curves of body weight change in the control group,maytansine group, and the groups treated with compounds 30 and 31 in theLXFE 937 squamous cell lung carcinoma model.

FIG. 9 shows tumor growth curves of the control group, maytansine group,and the groups treated with compounds 30 and 31 in the LXFA 737 lungadenocarcinoma model.

FIG. 10 shows curves of body weight change in the control group,maytansine group, and the groups treated with compounds 30 and 31 in theLXFA 737 lung adenocarcinoma model.

FIG. 11 shows tumor growth curves of the control group, maytansinegroup, and the groups treated with compounds 32, 30, and 31 in theMDA-MB 231 breast cancer model.

FIG. 12 shows curves of body weight change in the control group,maytansine group, and the groups treated with compounds 32, 30, and 31in the MDA-MB 231 breast cancer model.

FIG. 13 shows tumor growth curves of the control group, maytansinegroup, and the groups treated with compounds 30 and 31 in the A2780ovarian cancer model.

FIG. 14 shows curves of body weight change in the control group,maytansine group, and the groups treated with compounds 30 and 31 in theA2780 ovarian cancer model.

FIG. 15 shows tumor growth curves of the control group, maytansinegroup, and the groups treated with compounds 30 and 31 in the MDA-MB 468breast cancer model.

FIG. 16 shows curves of body weight change in the control group,maytansine group, and the groups treated with compounds 30 and 31 in theMDA-MB 468 breast cancer model.

DETAILED DESCRIPTION

Unless otherwise defined herein, scientific and technical terms used inthis application shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature relating totechniques of chemistry, molecular biology, cell and cancer biology,immunology, microbiology, pharmacology, and protein chemistry, describedherein, are those well-known and commonly used in the art.

All publications, patents and published patent applications referred toin this application are specifically incorporated by reference herein.In case of conflict, the present specification, including its specificdefinitions, will control. Unless otherwise specified, it is to beunderstood that each embodiment disclosed herein may be used alone or incombination with any one or more other embodiments of the invention.

Definitions

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer (or components) or group of integers (or components),but not the exclusion of any other integer (or components) or group ofintegers (or components).

Throughout the application, where a compound or composition is describedas having, including, or comprising, specific components, it iscontemplated that such compound or composition also may consistessentially of, or consist of, the recited components. Similarly, wheremethods or processes are described as having, including, or comprisingspecific process steps, the processes also may consist essentially of,or consist of, the recited processing steps. Further, it should beunderstood that the order of steps or order for performing certainactions is immaterial so long as the compounds, compositions and methodsdescribed herein remains operable. Moreover, two or more steps oractions can be conducted simultaneously.

The singular forms “a,” “an,” and “the” include the plurals unless thecontext clearly dictates otherwise.

The term “including” is used to mean “including but not limited to.”“Including” and “including but not limited to” are used interchangeably.

The term “or” as used herein should be understood to mean “and/or”,unless the context clearly indicates otherwise.

The terms “drug,” “agent,” “therapeutic agent”, “therapeutically activeagent”, “cytotoxic agent or drug”, “highly cytotoxic agent or drug”, or“therapeutically effective substance” are used to mean any compoundwhich brings about a pharmacological effect either by itself or afterits conversion in the organism in question, and thus also includes thederivatives from these conversions. The pharmacological effect of thedrugs of the composition according to the present disclosure can be asingle effect only, e.g. a cytostatic and/or cytotoxic effect, or abroad pharmacological spectrum of actions, such as an immunosuppressiveand antiphlogistic effect at the same time.

The terms “patient,” “subject,” or “individual” are used interchangeablyand refer to either a human or a non-human animal. These terms includemammals such as humans, primates, livestock animals (e.g., bovines,porcines), companion animals (e.g., canines, felines) and rodents (e.g.,mice and rats). In certain embodiments, the patient or subject is ahuman patient or subject, such as a human patient having a condition inneed of treatment.

The term “pharmaceutical composition” refers to a composition suitablefor pharmaceutical use in a subject animal, including humans andmammals, e.g., combined with one or more pharmaceutically acceptablecarriers, excipients or solvents. Such a composition may also containdiluents, fillers, salts, buffers, stabilizers, solubilizers,protectants and other materials well known in the art. In certainembodiments, a pharmaceutical composition encompasses a compositioncomprising the active ingredient(s), and the inert ingredient(s) thatmake up the excipient, carrier or diluent, as well as any product thatresults, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, thepharmaceutical compositions of the present disclosure encompass anycomposition made by admixing a compound of the disclosure and one ormore pharmaceutically acceptable excipient(s), carrier(s) and/ordiluent(s).

The term “pharmaceutically acceptable carrier” refers to a non-toxiccarrier that may be administered to a patient, together with atherapeutically effective substance disclosed herein, and which does notdestroy the pharmacological activity of the agent. The term “excipient”refers to an additive in a formulation or composition that is not apharmaceutically active ingredient. In certain embodiments, a“pharmaceutically acceptable” substance is suitable for use in contactwith cells, tissues or organs of animals or humans without excessivetoxicity, irritation, allergic response, immunogenicity or other adversereactions, in the amount used in the dosage form according to the dosingschedule, and commensurate with a reasonable benefit/risk ratio. Incertain embodiments, a “pharmaceutically acceptable” substance that is acomponent of a pharmaceutical composition is, in addition, compatiblewith the other ingredient(s) of the composition. In certain embodiments,the terms “pharmaceutically acceptable excipient”, “pharmaceuticallyacceptable carrier” and “pharmaceutically acceptable diluent” encompass,without limitation, pharmaceutically acceptable inactive ingredients,materials, compositions and vehicles, such as liquid fillers, solidfillers, diluents, excipients, carriers, solvents and encapsulatingmaterials. Carriers, diluents and excipients also include allpharmaceutically acceptable dispersion media, coatings, buffers,isotonic agents, stabilizers, absorption delaying agents, antimicrobialagents, antibacterial agents, antifungal agents, adjuvants, etc. Exceptinsofar as any conventional excipient, carrier or diluent isincompatible with the active ingredient; the present disclosureencompasses the use of conventional excipients, carriers and diluents inpharmaceutical compositions. See, e.g., Remington: The Science andPractice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins(Philadelphia, Pennsylvania, 2005); Handbook of PharmaceuticalExcipients, 5th Ed., Rowe et al., Eds., The Pharmaceutical Press and theAmerican Pharmaceutical Association (2005); Handbook of PharmaceuticalAdditives, 3rd Ed., Ash and Ash, Eds., Gower Publishing Co. (2007); andPharmaceutical Preformulation and Formulation, Gibson, Ed., CRC PressLLC (Boca Raton, Florida, 2004).

The terms “pharmaceutically effective amount,” “therapeuticallyeffective amount,” or “therapeutically effective dose” refer to anamount effective to treat a disease or condition in a patient, e.g.,effecting a beneficial and/or desirable alteration in the general healthof a patient suffering from a disease (e.g., cancer) or condition,treatment, healing, inhibition or amelioration of a physiologicalresponse or condition, etc. The full therapeutic effect does notnecessarily occur by administration of one dose, and may occur onlyafter administration of a series of doses. Thus, a therapeuticallyeffective amount may be administered in one or more administrations. Theprecise effective amount needed for a subject will depend upon, forexample, the subject's size, health and age, the nature and extent ofdisease, the therapeutics or combination of therapeutics selected foradministration, and the mode of administration. The skilled worker canreadily determine the effective amount for a given situation by routineexperimentation. The skilled worker will recognize that treating cancerincludes, but is not limited to, killing cancer cells, preventing thegrowth of new cancer cells, causing tumor regression (a decrease intumor size), causing a decrease in metastasis, improving vital functionsof a patient, improving the well-being of the patient, decreasing pain,improving appetite, improving the patient's weight, and any combinationthereof. The terms “pharmaceutically effective amount,” “therapeuticallyeffective amount,” or “therapeutically effective dose” also refer to theamount required to improve the clinical symptoms of a patient. Thetherapeutic methods or methods of treating cancer described herein arenot to be interpreted or otherwise limited to “curing” cancer.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition. As used herein, and as well understood in the art,“treatment” is an approach for obtaining beneficial or desired results,including clinical results. Beneficial or desired clinical results caninclude, but are not limited to, alleviation, amelioration, or slowingthe progression, of one or more symptoms or conditions associated with acondition, e.g., cancer, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Exemplary beneficialclinical results are described herein.

“Administering” or “administration of” a substance, a compound or anagent to a subject can be carried out using one of a variety of methodsknown to those skilled in the art. For example, a compound or an agentcan be administered, intravenously, arterially, intradermally,intramuscularly, intraperitoneally, subcutaneously, ocularly,sublingually, orally (by ingestion), intranasally (by inhalation),intraspinally, intracerebrally, and transdermally (by absorption, e.g.,through a skin duct). A compound or agent can also appropriately beintroduced by rechargeable or biodegradable polymeric devices or otherdevices, e.g., patches and pumps, or formulations, which provide for theextended, slow or controlled release of the compound or agent.Administering can also be performed, for example, once, a plurality oftimes, and/or over one or more extended periods. In some aspects, theadministration includes both direct administration, includingself-administration, and indirect administration, including the act ofprescribing a drug. For example, as used herein, a physician whoinstructs a patient to self-administer a drug, or to have the drugadministered by another and/or who provides a patient with aprescription for a drug is administering the drug to the patient. When amethod is part of a therapeutic regimen involving more than one agent ortreatment modality, the disclosure contemplates that the agents may beadministered at the same or differing times and via the same ordiffering routes of administration. Appropriate methods of administeringa substance, a compound or an agent to a subject will also depend, forexample, on the age of the subject, whether the subject is active orinactive at the time of administering, whether the subject iscognitively impaired at the time of administering, the extent of theimpairment, and the chemical and biological properties of the compoundor agent (e.g. solubility, digestibility, bioavailability, stability andtoxicity).

The term “substituted” refers to moieties having substituents replacinghydrogen on one or more carbons of the backbone of a chemical compound.It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc. As used herein, the term “substituted” iscontemplated to include all permissible substituents of organiccompounds. In a broad aspect, the permissible substituents includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and non-aromatic substituents of organiccompounds. The permissible substituents can be one or more and the sameor different for appropriate organic compounds. For purposes of thedisclosure, the heteroatoms such as nitrogen may have hydrogensubstituents, and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms.Substituents can include any substituents described herein, for example,a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, an alkylthio,an acyloxy, a phosphoryl, a phosphate, a phosphonate, an amino, anamido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl,an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic (e.g., C₆-C₁₂ aryl)or heteroaromatic (e.g., heteroaryl) moiety.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the application includesinstances where the circumstance occurs and instances where it does not.For example, the phrase “optionally substituted” means that anon-hydrogen substituent may or may not be present on a given atom, and,thus, the application includes structures wherein a non-hydrogensubstituent is present and structures wherein a non-hydrogen substituentis not present.

Unless specifically stated as “unsubstituted,” references to chemicalmoieties herein are understood to include substituted variants. Forexample, reference to an “alkyl” group or moiety implicitly includesboth substituted and unsubstituted variants. Examples of substituents onchemical moieties include but is not limited to, halogen, hydroxyl,carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl),thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl,alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido,amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate,sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, oraryl or heteroaryl moiety.

“Aryl” indicates an aromatic carbon ring having the indicated number ofcarbon atoms, for example, 6 to 12 or 6 to 10 carbon atoms, in the ring.Aryl groups may be monocyclic or polycyclic (e.g., bicyclic, tricyclic).In some instances, both rings of a polycyclic aryl group are aromatic(e.g., naphthyl). In other instances, polycyclic aryl groups may includea non-aromatic ring (e.g., cycloalkyl, cycloalkenyl, heterocycloalkyl,heterocycloalkenyl) fused to an aromatic ring, provided the polycyclicaryl group is bound to the parent structure via an atom in the aromaticring. Thus, a 1,2,3,4-tetrahydronaphthalen-5-yl group (wherein themoiety is bound to the parent structure via an aromatic carbon atom) isconsidered an aryl group, while 1,2,3,4-tetrahydronaphthalen-1-yl(wherein the moiety is bound to the parent structure via a non-aromaticcarbon atom) is not considered an aryl group. Similarly, a1,2,3,4-tetrahydroquinolin-8-yl group (wherein the moiety is bound tothe parent structure via an aromatic carbon atom) is considered an arylgroup, while 1,2,3,4-tetrahydroquinolin-1-yl group (wherein the moietyis bound to the parent structure via a non-aromatic nitrogen atom) isnot considered an aryl group. However, the term “aryl” does notencompass or overlap with “heteroaryl,” as defined herein, regardless ofthe point of attachment (e.g., both quinolin-5-yl and quinolin-2-yl areheteroaryl groups).

“Heteroaryl” indicates an aromatic ring containing the indicated numberof ring atoms (e.g., 5 to 12, or 5 to 10 membered heteroaryl) made up ofone or more heteroatoms (e.g., 1, 2, 3 or 4 heteroatoms) selected fromN, O and S and with the remaining ring atoms being carbon. 5-Memberedheteroaryl is a heteroaryl having 5 ring atoms. 6-Membered heteroaryl isa heteroaryl having 6 ring atoms. Heteroaryl groups do not containadjacent S and O atoms. In some embodiments, the total number of S and Oatoms in the heteroaryl group is not more than 2. In some embodiments,the total number of S and O atoms in the heteroaryl group is not morethan 1. Unless otherwise indicated, heteroaryl groups may be bound tothe parent structure by a carbon or nitrogen atom, as valency permits.For example, “pyridyl” includes 2-pyridyl, 3-pyridyl and 4-pyridylgroups, and “pyrrolyl” includes 1-pyrrolyl, 2-pyrrolyl and 3-pyrrolylgroups. When nitrogen is present in a heteroaryl ring, it may, where thenature of the adjacent atoms and groups permits, exist in an oxidizedstate (i.e., N⁻—O⁻). Additionally, when sulfur is present in aheteroaryl ring, it may, where the nature of the adjacent atoms andgroups permits, exist in an oxidized state (i.e., S⁺—O⁻ or SO₂).Heteroaryl groups may be monocyclic or polycyclic (e.g., bicyclic,tricyclic).

In some instances, a heteroaryl group is monocyclic. Examples includepyrrole, pyrazole, imidazole, triazole (e.g., 1,2,3-triazole,1,2,4-triazole, 1,3,4-triazole), tetrazole, furan, isoxazole, oxazole,oxadiazole (e.g., 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole),thiophene, isothiazole, thiazole, thiadiazole (e.g., 1,2,3-thiadiazole,1,2,4-thiadiazole, 1,3,4-thiadiazole), pyridine, pyridazine, pyrimidine,pyrazine, triazine (e.g., 1,2,4-triazine, 1,3,5-triazine) and tetrazine.

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbyl-C(O)—, e.g., alkyl-C(O)—.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, and branched-chain alkyl groups.In some embodiments, a straight chain or branched chain alkyl has 30 orfewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chains,C₄-C₃₀ for branched chains), and in other embodiments 20 or fewer. Incertain embodiments, alkyl groups are lower alkyl groups, e.g., methyl,ethyl, n-propyl, i-propyl, n-butyl and n-pentyl. Moreover, the term“alkyl” as used throughout the specification, examples, and claims isintended to include both “unsubstituted alkyls” and “substitutedalkyls”, the latter of which refers to alkyl moieties havingsubstituents replacing hydrogen on one or more carbons of thehydrocarbon backbone. In certain embodiments, a straight chain orbranched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g.,C₁-C₃₀ for straight chains, C₃-C₃₀ for branched chains). In someembodiments, the chain has ten or fewer carbon (C₁-C₁₀) atoms in itsbackbone. In other embodiments, the chain has six or fewer carbon(C₁-C₆) atoms in its backbone.

The terms “hydrazone moiety” or “hydrazone” refer to E and/or Zhydrazones, e.g.,

The stereochemistry of the hydrazone moiety can be E or Z. The termhydrazone as used herein includes both E and Z isomers. The hydrazonemoieties disclosed herein are generally drawn in one configuration, butit is understood that this disclosure can include both E and/or Z.

At various places in the present specification substituents of compoundsof the disclosure are disclosed in groups or in ranges. It isspecifically intended that the disclosure include each and everyindividual sub-combination of the members of such groups and ranges. Forexample, the term “C₁-C₆ alkyl” is specifically intended to discloseindividually methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,isobutyl, etc.

A “pharmaceutically acceptable salt” is a salt of a compound that issuitable for pharmaceutical use, including but not limited to metalsalts (e.g., sodium, potassium, magnesium, calcium, etc.), acid additionsalts (e.g., mineral acids, carboxylic acids, etc.), and base additionsalts (e.g., ammonia, organic amines, etc.). The acid addition salt formof a compound that occurs in its free form as a base can be obtained bytreating said free base form with an appropriate acid such as aninorganic acid, for example, a hydrohalic such as hydrochloric orhydrobromic, sulfuric, nitric, phosphoric and the like; or an organicacid, such as, for example, acetic, hydroxyacetic, propanoic, lactic,pyruvic, malonic, succinic, maleic, fumaric, malic, tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclic, salicylic, p-aminosalicylic, pamoic and the like (see, e.g., WO01/062726. Some pharmaceutically acceptable salts listed by Berge etal., Journal of Pharmaceutical Sciences, 66: 1-19 (1977), incorporatedherein by reference in its entirety). Compounds containing acidicprotons may be converted into their therapeutically active, non-toxicbase addition salt form, e.g. metal or amine salts, by treatment withappropriate organic and inorganic bases. Appropriate base salt formsinclude, for example, ammonium salts, alkali and earth alkaline metalsalts or ions, e. g., lithium, sodium, potassium, magnesium, calciumsalts and the like, salts with organic bases, e. g.N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids suchas, for example, arginine, lysine and the like. Conversely, said saltforms can be converted into the free forms by treatment with anappropriate base or acid. Compounds and their salts can be in the formof a solvate, which is included within the scope of the presentdisclosure. Such solvates include for example hydrates, alcoholates andthe like (see, e.g., WO 01/062726).

The disclosure further provides pharmaceutical compositions comprisingone or more compounds of the disclosure together with a pharmaceuticallyacceptable carrier or excipient. Compounds or pharmaceuticalcompositions of the disclosure may be used in vitro or in vivo.

The term “isomer” as used herein includes, but is not limited to,tautomers, cis- and trans-isomers (E (entgegen), Z (zusammen)), R- andS-enantiomers (said R and S notation is used in correspondence with therules described in Pure Appl. Chem. (1976), 45, 11-30), diastereomers,(D)-isomers, (L)-isomers, stereoisomers, the racemic mixtures thereof,and other mixtures thereof. All such isomers, as well as mixturesthereof, are intended to be included in this disclosure. Tautomers,while not explicitly indicated in the formulae described herein, areintended to be included within the scope of the present disclosure.

The disclosure further includes isotopically-labeled or enrichedcompounds of the disclosure. An “isotopically” or “radio-labeled”compound is a compound of the disclosure where one or more atoms arereplaced or substituted by an atom having an atomic mass or mass numberdifferent from the atomic mass or mass number typically found in nature(i.e., naturally occurring). Suitable radionuclides that may beincorporated in compounds of the present disclosure include but are notlimited to ²H (also written as D for deuterium), ³H (also written as Tfor tritium), ¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl,⁸²Br, ⁷⁵Br, ⁷⁶Br, and ⁷⁷Br. The radionuclide that is incorporated in theinstant radio-labeled compounds will depend on the specific applicationof that radio-labeled compound. For example, for in vitrometalloprotease labeling and competition assays, compounds thatincorporate ³H, ¹⁴C, ⁸²Br, ³⁵S or will generally be most useful. Forradio-imaging applications ¹¹C, ¹⁸F, ⁷⁵Br, ⁷⁶Br or ⁷⁷Br will generallybe most useful. Tritium (³H) and ¹⁴C may be useful for ADME studies. Insome embodiments, each alkyl, cycloalkyl, alkene, alkylene, and alkoxyis optionally substituted by one or more -D or —F.

Compounds of the Disclosure

Embodiments of the present disclosure provide a compound having thestructure represented by Formula (I):

-   -   or a pharmaceutically acceptable salt, hydrate, solvate, or        isomer thereof,    -   wherein:    -   R¹ is selected from —H and C₁-C₄ alkyl;    -   Spacer is selected from:

-   -   V is absent or selected from —CH₂—, —O— and —NR³—, wherein R³ is        —H or C₁-C₄ alkyl;    -   each R² is independently selected from —H, halogen (e.g., —F,        —Cl, —Br or —I) and C₁-C₄ alkyl or two R²s taken together form a        C₃-C₆, cycloalkyl;    -   n is 0-3;    -   X is absent or selected from —CH₂—, —O—, —S—, —Se—, and —NR⁴—,        wherein R⁴ is —H or C₁-C₄ alkyl;    -   Y is selected from ═CH— and ═N—;    -   Z¹, Z², Z³ and Z⁴ are each independently selected from —H,        halogen (e.g., —F, —Cl, —Br or —I), —CF₃, —OCH₃, —CN, —NO₂,        C₁-C₄ alkyl and C₂-C₄ alkoxy;    -   AA is an amino acid selected from glycine, D or L proline,        sarcosine, N-ethyl-glycine, D or L alanine, D or L        N-methylalanine, β-alanine, N-methyl-β-alanine,        α-aminoisobutyric acid, and N-methyl-α-aminoisobutyric acid;    -   R′ is selected from O and

-   -   Y′ is absent or selected from an optionally substituted C₁-C₆        alkyl, —NH—C(O)—, and —C(O)—NH—; or Y′ is selected from the        group consisting of:

wherein n=0-6;

-   -   R^(1′) is absent or selected from the group consisting of:

-   -   wherein M¹ is a pharmaceutically acceptable counter ion (e.g.,        H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, NR₄ ⁺, and NHR₃ ⁺; wherein R is H or        C₁-C₄ alkyl);    -   R² is optionally substituted C₁-C₁₈ alkyl wherein optionally up        to six carbon atoms in said Ct-Cis alkyl are each independently        replaced with —OCH₂CH₂—;    -   Z^(1′), Z^(2′), Z^(4′) and Z^(4′) are each independently        selected from —H, halogen (e.g., —F, —Cl, —Br or —I), —CF₃,        —OCH₃, —CN, —NO₂, —SO₃M², and C₁-C₄ alkyl wherein M² is a        pharmaceutically acceptable counter ion (e.g., H⁺, Na⁺, K⁺,        Ca²⁺, Mg²⁺, NR₄ ⁺, and NHR₃ ⁺; wherein R is H or C₁-C₄ alkyl);    -   TBG is a thiol-binding group selected from an optionally        substituted maleimide group, an optionally substituted        haloacetamide group, an optionally substituted haloacetate        group, an optionally substituted pyridylthio group, an        optionally substituted isothiocyanate group, an optionally        substituted vinylcarbonyl group, an optionally substituted        aziridine group, an optionally substituted disulfide group, an        optionally substituted acetylene group, and an optionally        substituted N-hydroxysuccininide ester group;    -   wherein said TBG is optionally bound to a thiol-bearing        macromolecular carrier or thiol-bearing tumor-specific carrier.

In some embodiments, in the compound of Formula (I), R¹ is selected from—H and C₁-C₄ alkyl;

-   -   Spacer is selected from:

-   -   V is absent or selected from —CH₂—, —O— and —NR³—, wherein R³ is        —H or C₁-C₄ alkyl;    -   each R² is independently selected from —H, halogen (e.g., —F,        —Cl, —Br or —I) and C₁-C₄ alkyl or two R²s taken together form a        C₃-C₆, cycloalkyl;    -   n is 0-3;    -   X is absent or selected from —CH₂—, —O—, —S—, —Se—, and —NR⁴—,        wherein R⁴ is —H or C₁-C₄ alkyl;    -   Y is selected from ═CH— and ═N—;    -   Z¹, Z², Z³ and Z⁴ are each independently selected from —H,        halogen (e.g., —F, —Cl, —Br or —I), —CF₃, —OCH₃, —CN, —NO₂,        C₁-C₄ alkyl and C₂-C₄ alkoxy;    -   AA is an amino acid selected from glycine, D or L proline,        sarcosine, D or L alanine, D or L N-methylalanine, β-alanine,        N-methyl-β-alanine, α-aminoisobutyric acid, and        N-methyl-α-aminoisobutyric acid;    -   R′ is selected from O and

Y′ is absent or selected from an optionally substituted C₁-C₆ alkyl,—NH—C(O)—, and —C(O)—NH—; or Y′ is selected from the group consistingof:

wherein n=0-6;

-   -   R^(1′) is absent or selected from the group consisting of:

-   -   wherein M¹ is a pharmaceutically acceptable counter ion (e.g.,        H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, NR₄ ⁺, and NHR₃ ⁺; wherein R is H or        C₁-C₄ alkyl);    -   R² is optionally substituted C₁-C₁₈ alkyl wherein optionally up        to six carbon atoms in said Ct-Cis alkyl are each independently        replaced with —OCH₂CH₂—;    -   Z^(1′), Z^(2′), Z^(3′) and Z^(4′) are each independently        selected from —H, halogen (e.g., —F, —Cl, —Br or —I), —CF₃,        —OCH₃, —CN, —NO₂, —SO₃M², and C₁-C₄ alkyl wherein M² is a        pharmaceutically acceptable counter ion (e.g., H⁺, Na⁺, K⁺,        Ca²⁺, Mg²⁺, NR₄ ⁺, and NHR₃ ⁺; wherein R is H or C₁-C₄ alkyl);    -   TBG is a thiol-binding group selected from an optionally        substituted maleimide group, an optionally substituted        haloacetamide group, an optionally substituted haloacetate        group, an optionally substituted pyridylthio group, an        optionally substituted isothiocyanate group, an optionally        substituted vinylcarbonyl group, an optionally substituted        aziridine group, an optionally substituted disulfide group, an        optionally substituted acetylene group, and an optionally        substituted N-hydroxysuccininide ester group;

wherein said TBG is optionally bound to a thiol-bearing macromolecularcarrier or thiol-bearing tumor-specific carrier.

In some embodiments, R′ is O. These novel compounds can represent activespecies, and may be, e.g., the active component of a drug deliverysystem or an active metabolite that is released from a drug deliverysystem.

In certain embodiments, the compound of Formula (I) where R′ is O has astructure of any one of Formulae (II′), (III′) and (IV′):

-   -   or a pharmaceutically acceptable salt, hydrate, solvate, or        isomer thereof, wherein:    -   each R² is independently selected from —H, and C₁-C₄ alkyl or        two R²s taken together form a C₃-C₆, cycloalkyl;    -   X is absent or selected from —CH₂—, —O—, —S— and —NR³—, wherein        R³ is —H or C₁-C₄ alkyl;    -   Z¹, Z², Z³ and Z⁴ are each independently selected from —H,        halogen (e.g., —F, —Cl, —Br or —I), —CF₃, —OCH₃, —NO₂ and —CH₃;

-   -   or a pharmaceutically acceptable salt, hydrate, solvate, or        isomer thereof,    -   wherein:    -   X is absent or selected from —CH₂— and —NH—;    -   Y is ═CH— or ═N—;    -   Z¹, Z², Z³ and Z³ are each independently selected from —H,        halogen (e.g., —F, —Cl, —Br or —I), —CF₃, —OCH₃, —NO₂ and —CH₃;    -   AA is an amino acid selected from glycine, D or L proline,        sarcosine, N-ethyl-glycine, D or L alanine, D or L        N-methylalanine, β-alanine, N-methyl-β-alanine,        α-aminoisobutyric acid, and N-methyl-α-aminoisobutyric acid.

In yet other embodiments, in the compounds of Formula (IV′):

-   -   X is absent or selected from —CH₂— and —NH—; Y is ═CH— or ═N—;    -   Z¹, Z², Z³ and Z³ are each independently selected from —H,        halogen (e.g., —F, —Cl, —Br or —I), —CF₃, —OCH₃, —NO₂ and —CH₃;    -   AA is an amino acid selected from glycine, D or L proline,        sarcosine, D or L alanine, D or L N-methylalanine, β-alanine,        N-methyl-β-alanine, α-aminoisobutyric acid, and        N-methyl-α-aminoisobutyric acid.

In some embodiments, the compound is selected from the followingspecific compounds:

or a pharmaceutically acceptable salt, hydrate, solvate, or isomerthereof.

Other embodiments include prodrugs, e.g., those represented by Formula(I) where R′ is:

These novel compounds can represent a drug delivery system whereby anactive metabolite is released selectively from a drug delivery system.These include, e.g., albumin-binding prodrugs.

In certain embodiments, these compounds have a structure of any one ofFormulae (II), (III), and (IV):

-   -   or a pharmaceutically acceptable salt, hydrate, solvate, or        isomer thereof, wherein:    -   each R² is independently selected from —H, and C₁-C₄ alkyl or        two R²s taken together form a C₃-C₆-cycloalkyl;    -   X is absent or selected from —CH₂—, —O—, —S— and —NR³—, wherein        R³ is —H or C₁-C₄ alkyl;    -   Z¹, Z², Z³ and Z⁴ are each independently selected from —H,        halogen (e.g., —F, —Cl, —Br or —I), —CF₃, —OCH₃, —NO₂ and —CH₃;

-   -   or a pharmaceutically acceptable salt, hydrate, solvate, or        isomer thereof,    -   wherein:    -   X is absent or selected from —CH₂— and —NH—;    -   Y is ═CH— or ═N—;    -   Z¹, Z², Z³ and Z³ are each independently selected from —H,        halogen (e.g., —F, —Cl, —Br or —I), —CF₃, —OCH₃, —NO₂ and —CH₃;    -   AA is an amino acid selected from glycine, D or L proline,        sarcosine, N-ethyl-glycine, D or L alanine, D or L        N-methylalanine, β-alanine, N-methyl-β-alanine,        α-aminoisobutyric acid, and N-methyl-α-aminoisobutyric acid; and    -   where R′ is:

In yet other embodiments, in the compounds of Formula (IV′):

-   -   X is absent or selected from —CH₂— and —NH—;    -   Y is ═CH— or ═N—;    -   Z¹, Z², Z³ and Z³ are each independently selected from —H,        halogen (e.g., —F, —Cl, —Br or —I), —CF₃, —OCH₃, —NO₂ and —CH₃;    -   AA is an amino acid selected from glycine, D or L proline,        sarcosine, D or L alanine, D or L N-methylalanine, β-alanine,        N-methyl-β-alanine, α-aminoisobutyric acid, and        N-methyl-α-aminoisobutyric acid; and    -   where R′ is:

In some embodiments, R¹ is —H. In other embodiments, at least one of Z¹,Z², Z³ and Z⁴ is not H and/or at least one of Z¹, Z², Z³ and Z⁴ is —F or—NO₂. In some embodiments, when n is 0, X is absent. In otherembodiments, when n is 0, X is —CH₂—. In some embodiments, n is 0 and Xis —O— or —S—. Additional embodiments include pharmaceuticallyacceptable salts, solvates, hydrates, tautomers, and solid forms of thedisclosed compounds.

In some embodiments, the compound is selected from the followingspecific compounds:

or a pharmaceutically acceptable salt, hydrate, solvate, or isomerthereof.

In certain embodiments, the compound is not bound to a thiol-bearingmacromolecular carrier or thiol-bearing tumor-specific carrier. In otherembodiments, the compound is bound to a thiol-bearing macromolecularcarrier or thiol-bearing tumor-specific carrier. For example, thethiol-bearing macromolecular carrier or thiol-bearing tumor-specificcarrier is selected from endogenous albumin, exogenous albumin, anantibody, an antibody fragment, a peptide, a natural or syntheticpolymer, a liposome and a nanoparticle.

In some embodiments, the TBG is an optionally substituted maleimidegroup, for example, an unsubstituted maleimide group. In someembodiments, the maleimide group binds rapidly and selectively to thecysteine-34 of albumin after administration to a subject, such as ahuman.

In some embodiments, Z^(1′) is selected from —NO₂ or —SO₃M² and/or andY′ is selected from —NHC(O)— or

In some embodiments, R^(1′) is

In some embodiments, R^(2′) is selected from optionally substitutedC₁-C₁₈ alkyl wherein optionally up to six carbon atoms in said C₁-C₁₈alkyl are each independently replaced with —OCH₂CH₂— (e.g., 1, 2, 3, 4,5 or 6 six carbon atoms are replaced with —OCH₂CH₂—).

In some embodiments, R′ is:

For example, R′ may be:

Specific compounds within the present disclosure include the following:

or a pharmaceutically acceptable salt, hydrate, solvate, or isomerthereof.

Pharmaceutical Compositions

In some embodiments, the disclosure provides a pharmaceuticalcomposition comprising a compound described herein. In some embodiments,the composition includes a compound of Formula (I) where R′ is:

or the various embodiments disclosed herein.

The total amount of a compound in a composition to be administered to apatient is one that is suitable for that patient. One of skill in theart would appreciate that different individuals may require differenttotal amounts of the therapeutically effective substance. In someembodiments, the amount of the compound is a pharmaceutically effectiveamount. The skilled worker would be able to determine the amount of thecompound in a composition needed to treat a patient based on factorssuch as, for example, the age, weight, and physical condition of thepatient. The concentration of the compound depends on its solubility inthe intravenous administration solution and the volume of fluid that canbe administered. For example, the concentration of the compound may befrom about 0.1 mg/ml to about 50 mg/ml in the injectable composition. Insome embodiments, the concentration of the compound may be in the rangeof about 0.1 mg/ml to about 40 mg/mL.

The pharmaceutical compositions and kits of the present disclosure mayalso contain diluents, fillers, salts, buffers, stabilizers,solubilizers, protectants and other materials well known in the art. Theterm “pharmaceutically acceptable” means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredient(s). The characteristics of the carrier will depend onthe route of administration.

The compositions may be administered in a variety of conventional ways.Exemplary routes of administration that can be used include oral,parenteral, intravenous, intra-arterial, cutaneous, subcutaneous,intramuscular, topical, intracranial, intraorbital, ophthalmic,intravitreal, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol, central nervoussystem (CNS) administration, or administration by suppository. In someembodiments, the compositions are suitable for parenteraladministration. These compositions may be administered, for example,intraperitoneally, intravenously, or intrathecally. In some embodiments,the compositions are injected intravenously. In some embodiments, areconstituted formulation can be prepared by reconstituting alyophilized compound composition in a reconstitution liquid comprisinge.g. an alcohol, DMSO, and/or polyethylene glycol and water and/or asalt buffer. Such reconstitution may comprise adding the reconstitutionliquid and mixing, for example, by swirling or vortexing the mixture.The reconstituted formulation then can be made suitable for injection bymixing e.g., Lactated Ringer's solution, 5% Glucose solution, isotonicsaline or a suitable salt buffer with the formulation to create aninjectable composition. One of skill in the art would appreciate that amethod of administering a therapeutically effective substanceformulation or composition would depend on factors such as the age,weight, and physical condition of the patient being treated, and thedisease or condition being treated. The skilled worker would, thus, beable to select a method of administration optimal for a patient on acase-by-case basis.

In some embodiments, the compounds and compositions disclosed herein arefor use in treating a cancer, a virus disease, autoimmune disease, acuteor chronic inflammatory disease, and a disease caused by bacteria,fungi, or other micro-organisms.

In some embodiments, the compound disclosed herein may be used in themanufacture of a medicament for treating a disease selected from acancer, a virus disease, autoimmune disease, acute or chronicinflammatory disease, and a disease caused by bacteria, fungi, or othermicro-organisms.

In some embodiments, the cancer is a blood cancer or a solid tumorcancer. In some embodiments, the cancer is selected from carcinoma,sarcoma, leukemia, lymphoma, multiple myeloma, and melanoma.

In some embodiments, the cancer is adenocarcinoma, uveal melanoma, acuteleukemia, acoustic neuroma, ampullary carcinoma, anal carcinoma,astrocytoma's, basalioma, pancreatic cancer, connective tissue tumor,bladder cancer, bronchial carcinoma, non-small cell bronchial carcinoma,breast cancer, Burkitt's lymphoma, corpus carcinoma, CUP syndrome, coloncancer, cancer of the small intestine, ovarian cancer, endometrialcarcinoma, gallbladder cancer, gallbladder carcinoma, uterine cancer,cervical cancer, neck, nose and ear tumors, hematological neoplasia's,hairy cell leukemia, urethral cancer, skin cancer, gliomas, testicularcancer, Kaposi's sarcoma, laryngeal cancer, bone cancer, colorectalcarcinoma, head/neck tumors, colon carcinoma, craniopharyngeoma, livercancer, leukemia, lung cancer, non-small cell lung cancer, Hodgkin'slymphoma, non-Hodgkin's lymphoma, stomach cancer, colon cancer,medulloblastoma, melanoma, meningioma, kidney cancer, renal cellcarcinomas, oligodendroglioma, esophageal carcinoma, osteolyticcarcinomas and osteoplastic carcinomas, osteosarcoma, ovarian carcinoma,pancreatic carcinoma, penile cancer, prostate cancer, tongue cancer,ovary carcinoma or lymph gland cancer.

In some embodiments, the present disclosure provides a kit comprising acompound as described herein and, a pharmaceutically acceptableexcipient, a carrier, and/or a diluent.

In some embodiments, one or more excipients may be included in thecomposition. One of skill in the art would appreciate that the choice ofany one excipient may influence the choice of any other excipient. Forexample, the choice of an excipient may preclude the use of one or moreadditional excipients because the combination of excipients wouldproduce undesirable effects. One of skill in the art would empiricallybe able to determine which excipients, if any, to include in thecompositions. Excipients may include, but are not limited to,co-solvents, solubilizing agents, buffers, pH adjusting agents, bulkingagents, surfactants, encapsulating agents, tonicity-adjusting agents,stabilizing agents, protectants, and viscosity modifiers. In someembodiments, it may be beneficial to include a pharmaceuticallyacceptable carrier in the compositions.

In some embodiments, a solubilizing agent may be included in thecompositions. Solubilizing agents may be useful for increasing thesolubility of any of the components of the composition, including acompound or an excipient. The solubilizing agents described herein arenot intended to constitute an exhaustive list, but are provided merelyas exemplary solubilizing agents that may be used in the compositions.In certain embodiments, solubilizing agents include, but are not limitedto, ethyl alcohol, tert-butyl alcohol, polyethylene glycol, glycerol,propylene glycol, methylparaben, propylparaben, polyethylene glycol,polyvinyl pyrrolidone, cyclodextrins such as dimethyl-β-cyclodextrin,hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, andtrimethyl-β-cyclodextrin, and combinations thereof, and anypharmaceutically acceptable salts and/or combinations thereof

The pH of the compositions may be any pH that provides desirableproperties for the formulation or composition. Desirable properties mayinclude, for example, compound stability, increased compound retentionas compared to compositions at other pH values, and improved filtrationefficiency. In some embodiments, the pH value of the compositions may befrom about 3.0 to about 9.0, e.g., from about 5.0 to about 7.0. Inparticular embodiments, the pH value of the compositions may be 5.5±0.1,5.6±0.1, 5.7±0.1, 5.8±0.1, 5.9±0.1, 6.0±0.1, 6.1±0.1, 6.2±0.1, 6.3±0.1,6.4±0.1, 6.5±0.1, 6.6±0.1, 6.7±0.1, 6.8±0.1, 6.9±0.1, 7.0±0.1, 7.1±0.1,and 7.2±0.1.

In some embodiments, it may be beneficial to buffer the pH by includingone or more buffers in the compositions. In certain embodiments, abuffer may have a pKa of, for example, about 5.5, about 6.0, or about6.5. One of skill in the art would appreciate that an appropriate buffermay be chosen for inclusion in compositions based on its pKa and otherproperties. Buffers are well known in the art. Accordingly, the buffersdescribed herein are not intended to constitute an exhaustive list, butare provided merely as exemplary buffers that may be used in theformulations or compositions of the present disclosure. In certainembodiments, a buffer includes, but is not limited to Tris, Tris-HCl,potassium phosphate, sodium phosphate, sodium citrate, sodium ascorbate,combinations of sodium and potassium phosphate, Tris/Tris-HCl, sodiumbicarbonate, arginine phosphate, arginine hydrochloride, histidinehydrochloride, cacodylate, succinate, 2-(N-morpholino)ethanesulfonicacid (MES), maleate, bis-tris, phosphate, carbonate, and anypharmaceutically acceptable salts and/or combinations thereof.

In some embodiments, a pH-adjusting agent may be included in thecompositions. Modifying the pH of a composition may have beneficialeffects on, for example, the stability or solubility of a compound, ormay be useful in making a composition suitable for parenteraladministration. pH-adjusting agents are well known in the art.Accordingly, the pH-adjusting agents described herein are not intendedto constitute an exhaustive list, but are provided merely as exemplarypH-adjusting agents that may be used in the compositions. pH-adjustingagents may include, for example, acids and bases. In some embodiments, apH-adjusting agent includes, but is not limited to, acetic acid,hydrochloric acid, phosphoric acid, sodium hydroxide, sodium carbonate,and combinations thereof.

In some embodiments, a bulking agent may be included in thecompositions. Bulking agents are commonly used in lyophilizedcompositions to provide added volume to the composition and to aidvisualization of the composition, especially in instances where thelyophilized pellet would otherwise be difficult to see. Bulking agentsalso may help prevent a blowout of the active component(s) of apharmaceutical composition and/or to aid cryoprotection of thecomposition. Bulking agents are well known in the art. Accordingly, thebulking agents described herein are not intended to constitute anexhaustive list, but are provided merely as exemplary bulking agentsthat may be used in the compositions.

Exemplary bulking agents may include carbohydrates, monosaccharides,disaccharides, polysaccharides, sugar alcohols, amino acids, and sugaracids, and combinations thereof. Carbohydrate bulking agents include,but are not limited to, mono-, di-, or poly-carbohydrates, starches,aldoses, ketoses, amino sugars, glyceraldehyde, arabinose, lyxose,pentose, ribose, xylose, galactose, glucose, hexose, idose, mannose,talose, heptose, glucose, fructose, methyl α-D-glucopyranoside, maltose,lactone, sorbose, erythrose, threose, arabinose, allose, altrose,gulose, idose, talose, erythrulose, ribulose, xylulose, psicose,tagatose, glucosamine, galactosamine, arabinans, fructans, fucans,galactans, galacturonans, glucans, mannans, xylans, inulin, levan,fucoidan, carrageenan, galactocarolose, pectins, amylose, pullulan,glycogen, amylopectin, cellulose, pustulan, chitin, agarose, keratin,chondroitin, dermatan, hyaluronic acid, xanthin gum, sucrose, trehalose,dextran, and lactose. Sugar alcohol bulking agents include, but are notlimited to, alditols, inositols, sorbitol, and mannitol. Sugar acidbulking agents include, but are not limited to, aldonic acids, uronicacids, aldaric acids, gluconic acid, isoascorbic acid, ascorbic acid,glucaric acid, glucuronic acid, gluconic acid, glucaric acid,galacturonic acid, mannuronic acid, neuraminic acid, pectic acids, andalginic acid. Amino acid bulking agents include, but are not limited to,glycine, histidine, and proline.

In some embodiments, a surfactant may be included in the compositions.Surfactants, in general, reduce the surface tension of a liquidcomposition. This may provide beneficial properties such as improvedease of filtration. Surfactants also may act as emulsifying agentsand/or solubilizing agents. Surfactants are well known in the art.Accordingly, the surfactants described herein are not intended toconstitute an exhaustive list, but are provided merely as exemplarysurfactants that may be used in the formulations or compositions of thepresent disclosure. Surfactants that may be included include, but arenot limited to, sorbitan esters such as polysorbates (e.g., polysorbate20 and polysorbate 80), lipopolysaccharides, polyethylene glycols (e.g.,PEG 400 and PEG 3000), poloxamers (i.e., pluronics), ethylene oxides andpolyethylene oxides (e.g., Triton X-100), saponins, phospholipids (e.g.,lecithin), and combinations thereof.

In some embodiments, an encapsulating agent may be included in thecompositions. Encapsulating agents can sequester molecules and helpstabilize or solubilize them. Encapsulating agents are well known in theart. Accordingly, the encapsulating agents described herein are notintended to constitute an exhaustive list, but are provided merely asexemplary encapsulating agents that may be used in the compositions.Encapsulating agents that may be included in compositions include, butare not limited to α-cyclodextrins, β-cyclodextrins, γ-cyclodextrin andcombinations thereof (e.g., α-cyclodextrin, dimethyl-α-cyclodextrin,hydroxyethyl-α-cyclodextrin, hydroxypropyl-α-cyclodextrin,trimethyl-α-cyclodextrin, p-cyclodextrin, dimethyl-p-cyclodextrin,hydroxyethyl-p-cyclodextrin, hydroxypropyl-p-cyclodextrin,trimethyl-p-cyclodextrin, Y-cyclodextrin, dimethyl-γ-cyclodextrin,hydroxyethyl-γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin,trimethyl-γ-cyclodextrin, and combinations thereof.

In some embodiments, a tonicity-adjusting agent may be included in thecompositions. The tonicity of a liquid composition is an importantconsideration when administering the composition to a patient, forexample, by parenteral administration. Tonicity-adjusting agents, thus,may be used to help make a composition suitable for administration.Tonicity-adjusting agents are well known in the art. Accordingly, thetonicity-adjusting agents described herein are not intended toconstitute an exhaustive list, but are provided merely as exemplarytonicity-adjusting agents that may be used in the compositions.Tonicity-adjusting agents may be ionic or non-ionic and include, but arenot limited to, inorganic salts, amino acids, carbohydrates, sugars,sugar alcohols, and carbohydrates. Exemplary inorganic salts may includesodium chloride, potassium chloride, sodium sulfate, and potassiumsulfate. An exemplary amino acid is glycine. Exemplary sugars mayinclude sugar alcohols such as glycerol, propylene glycol, glucose,sucrose, lactose, dextrose, and mannitol.

In some embodiments, a stabilizing agent may be included in thecompositions. Stabilizing agents help increase the stability of acompound in the compositions. This may occur by, for example, reducingdegradation or preventing aggregation of a compound. Without wishing tobe bound by theory, mechanisms for enhancing stability may includesequestration of the compound from a solvent or inhibiting free radicaloxidation of the therapeutically effective substance. Stabilizing agentsare well known in the art. Accordingly, the stabilizing agents describedherein are not intended to constitute an exhaustive list, but areprovided merely as exemplary stabilizing agents that may be used in thecompositions. Stabilizing agents may include, but are not limited to,emulsifiers and surfactants.

In some embodiments, a protectant may be included in the compositions.Protectants are agents that protect a pharmaceutically active ingredient(e.g., a therapeutically effective substance or compound) from anundesirable condition (e.g., instability caused by freezing orlyophilization, or oxidation). Protectants can include, for example,cryoprotectants, lyoprotectants, and antioxidants. Cryoprotectants areuseful in preventing loss of potency of an active pharmaceuticalingredient (e.g., an anthracycline compound) when a composition isexposed to a temperature below its freezing point. For example, acryoprotectant could be included in a reconstituted lyophilizedformulation so that the formulation could be frozen before dilution forintravenous administration. Cryoprotectants are well known in the art.Accordingly, the cryoprotectants described herein are not intended toconstitute an exhaustive list, but are provided merely as exemplarycryoprotectants that may be used in the compositions. Cryoprotectantsinclude, but are not limited to, solvents, surfactants, encapsulatingagents, stabilizing agents, viscosity modifiers, and combinationsthereof. Cryoprotectants may include, for example, disaccharides (e.g.,sucrose, lactose, maltose, and trehalose), polyols (e.g., glycerol,mannitol, sorbitol, and dulcitol), glycols (e.g., ethylene glycol,polyethylene glycol and propylene glycol).

Lyoprotectants are useful in stabilizing the components of a compositionsubjected to lyophilization. For example, a therapeutically effectivesubstance could be lyophilized with a lyoprotectant prior toreconstitution. Lyoprotectants are well known in the art. Accordingly,the lyoprotectants described herein are not intended to constitute anexhaustive list, but are provided merely as exemplary lyoprotectantsthat may be used in the compositions. Lyoprotectants include, but arenot limited to, solvents, surfactants, encapsulating agents, stabilizingagents, viscosity modifiers, and combinations thereof. Exemplarylyoprotectants may be, for example, sugars and polyols. Trehalose,sucrose, dextran, and hydroxypropyl-beta-cyclodextrin are non-limitingexamples of lyoprotectants.

Antioxidants are useful in preventing oxidation of the components of acomposition. Oxidation may result in aggregation of a drug product orother detrimental effects to the purity of the drug product or itspotency. Antioxidants are well known in the art. Accordingly, theantioxidants described herein are not intended to constitute anexhaustive list, but are provided merely as exemplary antioxidants thatmay be used in the compositions. Antioxidants may be, for example,sodium ascorbate, citrate, thiols, metabisulfite, and combinationsthereof.

In some embodiments, a viscosity-modifying agent may be included in thecomposition. Viscosity modifiers change the viscosity of liquidcompositions. This may be beneficial because viscosity plays animportant role in the ease with which a liquid composition is filtered.A composition may be filtered prior to lyophilization andreconstitution, or after reconstitution. Viscosity modifiers are wellknown in the art. Accordingly, the viscosity modifiers described hereinare not intended to constitute an exhaustive list, but are providedmerely as exemplary viscosity modifiers that may be used in thecompositions. Viscosity modifiers include solvents, solubilizing agents,surfactants, and encapsulating agents. Exemplary viscosity modifiersthat may be included in compositions include, but are not limited to,N-acetyl-DL-tryptophan and N-acetyl-cysteine.

Antitumor Activity in Human Tumor Xenograft Mice Models

The albumin-binding maytansinoids 30 and 31 demonstrated exceptionalantitumor activity in five human tumor xenograft models in nude miceinducing partial and complete tumor regressions in all human tumorxenograft evaluated (see FIGS. 3-16 ). This included starting tumorvolumes in the range of approximately 80-110 mm³ but also initialstarting tumor volumes of up to approximately 400 mm³. Furthermore, inmost cases therapy with albumin-binding maytansinoids 30 and 31 inducedlong-term remissions and a decrease in Relative Tumor Volume (RTV). Theparent compound maytansine was principally inactive in the tested modelsor only showed marginal tumor inhibition. Experimental procedure and theresults in the tumor-bearing mice models are described in detail inExamples 11-19 and FIGS. 3-16 .

Methods of Treatment

The compounds and compositions described herein are useful for a varietyof clinical applications. In embodiments, the methods of treatmentutilize a compound of composition that includes a compound of Formula(I) where R′ is:

or the various embodiments disclosed herein.

The compounds and compositions described herein can induce prolonged orlong-term inhibition of tumor growth. In certain embodiments, theprolonged or long-term inhibition of tumor growth is without any loss inbody weight or any or merely marginal bone marrow toxicity.

In some embodiments, the present disclosure provides a method fortreating a malignant disease comprising administering to a patient inneed thereof a therapeutically effective amount of a pharmaceuticalcomposition containing a compound described herein. For example, someembodiments include a method for treating a patient suffering from adisease or condition selected from a cancer, a virus disease, autoimmunedisease, acute or chronic inflammatory disease, and a disease caused bybacteria, fungi, and other micro-organisms, comprising administering tothe patient in need thereof a therapeutically effective amount of acompound according to the present disclosure.

The disclosure provides for methods of treating a condition or diseasein a patient, said condition or disease selected from a cancer, a virusdisease, autoimmune disease, acute or chronic inflammatory disease, anda disease caused by bacteria, fungi, or other micro-organisms,comprising administering to the patient a compound or a pharmaceuticalcomposition as described herein.

In some embodiments, the cancer comprises a vascularized tumor. In someembodiments, the cancer is a blood cancer or a solid tumor cancer. Insome embodiments, the cancer is selected from carcinoma, sarcoma,leukemia, lymphoma, multiple myeloma, and melanoma.

In some embodiments, the cancer is selected from adenocarcinoma, uvealmelanoma, acute leukemia, acoustic neuroma, ampullary carcinoma, analcarcinoma, astrocytoma, basalioma, pancreatic cancer, connective tissuetumor, bladder cancer, bronchial carcinoma, non-small cell bronchialcarcinoma, breast cancer, Burkitt's lymphoma, corpus carcinoma, CUPsyndrome, colon cancer, cancer of the small intestine, ovarian cancer,endometrial carcinoma, gallbladder cancer, uterine cancer, cervicalcancer, neck, nose and ear tumors, hematological neoplasia, hairy cellleukemia, urethral cancer, skin cancer, gliomas, testicular cancer,Kaposi's sarcoma, laryngeal cancer, bone cancer, colorectal carcinoma,head/neck tumors, colon carcinoma, craniopharyngeoma, liver cancer,leukemia, lung cancer, non-small cell lung cancer, Hodgkin's lymphoma,non-Hodgkin's lymphoma, stomach cancer, colon cancer, medulloblastoma,melanoma, meningioma, kidney cancer, renal cell carcinomas,oligodendroglioma, esophageal carcinoma, osteolytic carcinomas andosteoplastic carcinomas, osteosarcoma, ovarian carcinoma, pancreaticcarcinoma, penile cancer, prostate cancer, tongue cancer, ovarycarcinoma, and lymph gland cancer.

Some embodiments include a method of increasing the concentration of ametabolite of a compound in a tumor, comprising administering thecompound according to the present disclosure. In embodiments thecompound is a compound of Formula (I) where R′ is:

or the various embodiments disclosed herein. In some embodiments, theincrease is compared to an equivalent dose of the unmodified activeagent, e.g., the “unmodified active agent” may be the same compound ofFormula (I) where R′ is O.

Some embodiments include a method of reducing cytotoxicity of a compoundcomprising administering a compound or a pharmaceutical composition ofthe disclosure to a patient in need thereof, wherein the administrationresults in a reduction in cytotoxicity when compared to an equivalentdose of the unmodified active agent. For example, in some embodiments,the method of reducing cytotoxicity comprises administering a compoundof Formula (I) where R′ is:

or the various embodiments disclosed herein, and the “unmodified activeagent” is the same compound of Formula (I) where R′ is O.

EXEMPLIFICATION

With aspects of the present disclosure now being generally described,these will be more readily understood by reference to the followingexamples, which are included merely for purposes of illustration ofcertain features and embodiments of the present disclosure and are notintended to be limiting.

EQUIVALENTS

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, numerous equivalents to thecompounds, compositions, and methods of use thereof described herein.Such equivalents are considered to be within the scope of the presentdisclosure.

EXAMPLES

The following examples demonstrate the various embodiments and theaspects of the invention.

Abbreviations

The following is a list of abbreviations used in the Examples, withtheir full chemical names. If not defined, the terms have theirgenerally accepted meanings.

-   -   aq.=aqueous    -   Boc=N-tert-butoxycarbonyl    -   calcd.=calculated    -   DCM=dichloromethane    -   DIC=N,N′-diisopropylcarbodiimide    -   DMAP=N,N-dimethyl-4-aminopyridine    -   DMF=N,N-dimethylformamide    -   DMSO=dimethylsulfoxide    -   CV=column volume    -   EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide    -   eq=equivalents    -   ESI=electrospray ionization    -   Fmoc=fluorenylmethyloxycarbonyl    -   HATU=(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium        3-oxid hexafluorophosphate)    -   HOAt=1-hydroxy-7-azabenzotriazole    -   HOBt=N-hydroxybenzotriazole    -   HPLC=high-performance liquid chromatography    -   HRMS=high resolution mass spectrometry    -   HSA=human serum albumin    -   IC=ion chromatography    -   LC-MS=liquid chromatography mass spectroscopy    -   LRMS=low resolution mass spectrometry    -   MeCN=acetonitrile    -   MeOH=methanol    -   NM/FR=nuclear magnetic resonance spectroscopy    -   NP=normal phase    -   Np=4-nitrophenoxycarbonyl    -   nd=not detectable    -   na=not available    -   PBS=phosphate buffered saline, pH 7    -   RP=reverse phase    -   TFA=trifluoroacetic acid    -   TRIS=2-amino-2-(hydroxymethyl)-1,3-propanediol

Example 1 Preparation of Linker 1

Linker 1 may be prepared as described below and shown in Scheme 1.

Synthesis of 4-nitro-2-sulfobenzoic acid (A)

To a stirred solution of potassium permanganate (72 g, 460 mmol, 4.5 eq)in water (450 mL) was added within 10 s a solution of 4-nitro-2-sulfonicacid hydrate (26 g, 102 mmol, 1.0 eq) in Millipore water (100 mL). Theresulting purple mixture was stirred at 115° C. for 5 h and turned brownafter this time. HPLC analysis (PDA 220 nm) confirmed that the reactionwas finished after 5 h (>90% conversion). The reaction mixture wascooled down to room temperature. The brown solid formed during thereaction was removed through suction filtration on a Celite pad, washedwith Millipore water (300 mL) and the brown/yellow filtrate solution wasconcentrated to ca. 125 mL, with a rotary evaporator at 40° C.,acidified slowly with a 5 M HCl (ca. 2 mL) solution until a whitesuspension was formed (ca. pH 1.0). The white suspension was then heatedat 100° C. until a clear solution was obtained which was left to standin an ice bath for 10 min, until a white solid formed. The white solidwas obtained by suction filtration using a fritted filter. The whitesolid was then dried under high vacuum to give 4-nitro-2-sulfobenzoicacid. Yield: 18 g (72%). Purity by RP-HPLC, 220 nm, >95%. LRMS-ESI (m/z)calcd. for C₇H₄NO₇S [M−H]⁻: 245.98. Found: 245.83.

Synthesis of 4-amino-2-sulfobenzoic acid (B)

A stirred suspension of 4-nitro-2-sulfobenzoic acid (13 g, 51 mmol, 1.0eq) in water (75 mL) was heated at reflux until complete dissolution ofthe 4-nitro-2-sulfobenzoic acid. At that temperature was then addedacetic acid (7.2 mL) followed by iron powder (9.5 g, 180 mmol, 3.5 eq)that was added portion wise (˜1 g/min) over 10 min to avoid exothermicreaction. The reaction mixture was then left stirring under reflux for 1h. During this time, a brown solid formed and HPLC analysis (PDA 220 nm)confirmed that the reaction was finished (>95% conversion). The brownsolid was removed by suction filtration directly on a Celite pad (whenstill hot) and was further washed with hot water. The filtrate wasre-filtered. The resulting filtrate was concentrated with a rotaryevaporator at 40° C. to a final volume of 100 mL. Concentrated HCl wasadded dropwise until pH 1 was reached, and a white/yellow solidprecipitated. The suspension was left at 4° C. for 1 h. The solid wascollected by suction filtration using a fritted filter and was driedunder high vacuum to afford 4-amino-2-sulfobenzoic acid as a whitesolid. Yield: 9 g (81%). Purity by RP-HPLC, 220 nm, >95%. LRMS-ESI (m/z)calcd. for C₇H₄NO₅S [M−H]⁻: 216.00. Found: 216.16.

Synthesis of 6-maleimidohexanoyl chloride or EAMC-Cl (C)

To a stirring yellow solution of 6-maleimido caproic acid (EMC) (33 g,156 mmol, 1.0 eq) in dry DCM (150 mL) at room temperature and under N₂atmosphere, was added within 30 min (˜0.5 mL/min) oxalyl chloride (15mL, 171 mmol, 1.1 eq) using a dropping funnel. The reaction was stirredat room temperature for 5 h. The color of the reaction solution changedto dark yellow during the reaction time and HPLC analysis (PDA 220 nm)confirmed that the reaction was finished after 5 h (>95% conversion).Solvent was removed with a rotary evaporator at 40° C. to obtain an oil.This residual oil was dried under high vacuum overnight (solidifiedovernight). The obtained light brownish solid was crushed and dried forfurther 20 h under high vacuum to give 6-maleimidohexanoyl chloride as ayellow microcrystalline solid. The compound was used in the nextreaction without further purification. Yield: 34 g (95%). Purity byRP-HPLC, 220 nm, >95% as the methyl ester. LRMS-ESI (m/z) calcd. forC₁₁H₁₆NO₄ (as methyl ester) [M+H]⁺: 226.10. Found: 225.97.

Synthesis of 4-(6-maleimidohexanamido)-2-sulfobenzoic acid (D)

4-Amino-2-sulfobenzoic acid (18.5 g, 85.0 mmol, 1.0 eq) was dissolved inanhydrous DMF (300 mL) under N₂ atmosphere. The solution was cooled downto 4° C., and left stirring for 10 min. Then, 4-N-methylmorpholine (18.7mL, 170 mmol, 2.0 eq) was added dropwise (˜0.3 mL/min), within 1 h usinga dropping funnel to the cooled solution. To this dark brown mixture wasadded dropwise (˜0.5 g/min), within 1 h using a dropping funnel, asolution of EMC-Cl (29.3 g, 127 mmol, 1.5 eq) in anhydrous DMF (200 mL).The reaction mixture was stirred overnight and then allowed to reachroom temperature over 10 h. After completion of the reaction asindicated by HPLC analysis (PDA 220 nm, >95% conversion), the reactionsolution was dispensed in 8×50 mL falcon tubes.

The samples were centrifuged for 20 minutes at 10° C. and 4.000 rpm. Thesupernatants were removed by decantation, and the solids werere-suspended in 10 mL of DMF per each tube and centrifuged again for 20min at 10° C. and 4.000 rpm. All the DMF supernatants were combined andconcentrated under reduced pressure at 50° C. for 3 h to obtain a lightorange solid. The solid was re-suspended in methanol (250 mL) andtransferred to 8×50 mL falcon tubes. The samples were centrifuged for 20minutes at 10° C. and 4.000 rpm. The supernatants were removed bydecantation, and the solids were re-suspended in 5 mL of methanol pereach tube and centrifuged again for 20 min at 10° C. and 4.000 rpm. Allthe solids were combined and dried under high vacuum for 24 h to obtaina crystalline yellow solid. Yield: 17 g (48%). Purity by RP-HPLC reversephase, 220 nm, 80%. LRMS-ESI (m/z) calcd. for C₁₇H₁₇N₂O₈S [M−H]⁻:409.08. Found: 409.13.

Synthesis of2-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-5-(6-maleimidohexanamido)benzenesulfonicacid or Boc-protected linker 1 (E)

To a solution of 4-(6-maleimidohexanamido)-2-sulfobenzoic acid (17.0 g,41.4 mmol, 1.0 eq) in anhydrous DMF (350 mL) under N₂ atmosphere wereadded EDC-HCl (8.72 g, 45.5 mmol, 1.1 eq) and 1HOBt (6.15 g, 45.5 mmol,1.1 eq). The reaction mixture was left to stir 30 min at roomtemperature, and then tert-butyl-carbazate (7.12 g, 53.9 mmol, 1.3 eq)was added and the solution turns from clear yellow to reddish. Thereaction mixture was stirred at room temperature overnight. After thistime, completion of the reaction was confirmed by HPLC (PDA 220 nm, >95%conversion). The solvent was removed with a rotary evaporator at 40° C.and under high vacuum for 1 h, to afford purple-brown oil, which waspurified with a Biotage Isolera One flash purification System, with twopre-packed SNAP ULTRA 340 g cartridge, with Biotage® HP-Sphere™spherical silica. The tubes containing the desired product were combinedand dried with a rotary evaporator and under high vacuum for 10 h toobtain2-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-5-(6-maleimidohexanamido)benzenesulfonicacid as a foamy yellow solid. Yield: 9 g (42%). RP-HPLC (220 nm)>95%.LRMS-ESI (n z) calcd. for C₂₂H₂₇N₄O₉S [M−H]⁻: 523.16. Found: 523.15.

Synthesis of Linker 1

To a cooled (4-5° C.) solution of Boc-protected Linker 1 (10.2 g, 19.4mmol, 1.0 eq) in anhydrous DCM (30 mL) was added dropwise (˜0.5 mL/min)TFA (15 mL) within 30 min. After the addition, the cold bath wasremoved, and the reaction mixture was left to stir at room temperaturefor 3 h. After this time, completion of the reaction was confirmed byHPLC analysis (PDA 220 nm). The reaction mixture was poured dropwise insix falcon tubes, each of them with ca. 35 mL cold diethyl ether. Awhite precipitate formed immediately. The tubes were left at 4° C. for 3h. After centrifugation of the falcon tubes (4000 rpm, 20 min, 10° C.),the supernatants were removed by decantation and the solids werere-suspended in 5 mL of diethyl ether per each tube and centrifugedagain (4000 rpm, 20 min, 10° C.). The supernatants were removed again bydecantation and the solids collected and dried under high vacuum toafford the Linker 1 as white microcrystalline solid as a TFA salt.Yield: 10 g (96%). RP-HPLC (220 nm)>95%. LRMS-ESI (m/z) calcd. forC₁₇H₂₁N₄O₇S [M+H]⁺: 425.11. Found: 425.07. LRMS-ESI (m/z) calcd. forC₁₇H₁₉N₄O₇S [M−H]⁻: 423.11. Found: 423.12. HRMS-ESI (m/z) calcd. forC₁₇H₂₁N₄O₇S [M+H]⁺: 425.1125. Found: 425.1125. HRMS-ESI (m/z) calcd. forC₁₇H₁₉N₄O₇S [M−H]⁻: 423.0978. Found: 423.0980.

The structure was confirmed by ¹H NMR and ¹³C NMR: ¹H NMR (400 MHz,DMSO-d₆) δ 11.94 (s, 1H; C1-NH), 10.27 (s, 1H; C8-NH), 8.01 (d, J=2.2Hz, 1H; C4-CH), 7.93 (dd, J=8.5, 2.2 Hz, 1H; C6-CH), 7.68 (d, J=8.4 Hz,1H; C7-CH), 7.00 (s, 2H; C15-CH, C16-CH), 3.40 (t, J=7.0 Hz, 2H;C13-CH₂), 2.32 (t, J=7.4 Hz, 2H; C9-CH₂), 1.60 (p, J=7.5 Hz, 2H;C10-CH₂), 1.52 (p, J=7.2 Hz, 2H; C12-CH₂), 1.26 (q, J=8.8 Hz, 2H;C11-CH₂); ³C NMR (101 MHz, DMSO-d₆) δ 172.20 (C8), 171.54 (C14, C17),167.59 (C1), 145.73 (C5), 142.09 (C3), 134.90 (C15, C16), 132.09 (C7),123.79 (C2), 119.44 (C6), 117.47 (C4), 37.42 (C13), 36.65 (C9), 28.22(C12), 26.21 (C11), 24.90 (C10). Anal. caled. for C17H₂₁N₄O₇S·_(1/2)TFAC, 45.71; H, 4.26; N, 11.85; S, 6.78. Found: C, 46.2917; H, 4.4836; N,12.8879; S, 6.7886. TFA content 0.51%.

Linker 1 may also be prepared as described below and shown in Scheme 2.

Synthesis of5-amino-2-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)benzenesulfonicacid (F)

To a suspension of B (30.00 g, 138.12 mmol, 1.00 equiv.) in anhydrousacetonitrile (600 mL) was added triethylamine (41.93 g, 57.76 mL, 414.37mmol, 3.00 equiv.) and the mixture was stirred for 10 min. Afterwards,tert-butyl carbazate (27.38 g, 207.19 mmol, 1.50 equiv.) was added andthe mixture was cooled to −35° C. At this temperature, propylphosphonicanhydride solution, T3P, (114.27 g, 106.79 mL, 179.56 mmol, 50% sol. inethyl acetate, 1.3 equiv.) was added dropwise over 1 h. The reaction wasstirred at −35° C. for 2 h. The mixture was allowed to warm up to roomtemperature and filtered through Celite® 545 (100 g). Celite® wasadditionally washed with acetonitrile (500 mL). Both filtrates werecombined and concentrated to 250 mL. The solution was split equally into6 portions and the solvent was removed under reduced pressure. Eachportion was dissolved in dichloromethane containing 1% Et₃N (50 mL) andpurified by NP flash chromatography on a Biotage Isolera™ One FlashPurification System, with a pre-packed SNAP Ultra 340 g column, using astep gradient from 2% to 12% methanol (containing 1% NEt₃) in DCM(containing 1% NEt₃) over 7 column volumes. Then, the purified fractionsfrom all portions were combined, the solvent was removed under reducedpressure and the solid was dried under high vacuum to give titlecompound F as an off-white solid. Yield: 53.25 g, 108.0 mmol, 78.2% (NMRin DMSO-d6 showed the presence of 1.6 eq. triethylamine). HPLC (method9, 220 nm)>99%. LRMS-ESI (m/z) calcd. for C₁₂H₁₆N₃O₆S [M−H]⁻: 330.08.Found: 330.08.

Synthesis of N-ethyl-N-isopropylpropan-2-aminium2-(2-(tert-butoxy-carbonyl)hydrazine-1-carbonyl)-5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-)hexananido)benzenesulfonate(E)

To a mixture of F (45.00 g, 91.24 mmol, 1.00 equiv.) and6-maleimidocaproic acid (19.27 g, 91.24 mmol, 1.00 equiv.) was added,acetonitrile (450 mL), triethylamine (13.85 g, 19.08 mL, 136.86 mmol)and T3P (43.55 g, 40.70 mL, 136.86 mmol, 50% sol. in ethyl acetate) wereadded in one portion at room temperature. The solution was stirred atroom temperature for 24 h. The solvent was removed under reducedpressure. The crude was then purified by flash purification system usingseven pre-packed SNAP Ultra 340 g cartridge running a linear gradientfrom 2% methanol to 15% methanol in dichloromethane to give the titlecompound E as an off-white solid. Yield: 30.55 g, 53.5% (NMR in DMSO-d6showed the presence of 1.1 eq. triethylamine). HPLC (method 9, 220nm)>99%. LRMS-ESI (m/z) calcd. for C₂₂H₂₇N₄O₉S [M−H]⁻: 523.15. Found:523.26.

Synthesis of5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-2-(hydrazine-carbonyl)benzenesulfonicacid (Linker 1)

To a cold suspension (4° C.) of E, (10.00 g, 15.98 mmol, 1.00 equiv.) indichloromethane (50 mL) was added trifluoroacetic acid (18.22 g, 12.31mL, 159.81 mmol, 10.17 equiv.) over 15 min. The mixture was furtherstirred at 4° C. for 15 minutes, and then allowed to warm gradually toroom temperature and stirred for 150 min. The reaction mixture was addeddropwise via a separating funnel to a stirred solution of methyltert-butyl ether, MTBE, (400 mL) and dichloromethane (200 mL). Theresulted white solid was filtered through a 4 Å porosity fritted funneland washed sequentially with dichloromethane (2×150 mL) and MTBE (1×50mL), MeOH (1×50 mL) and again MTBE (2×150 mL). The solid was left to dryon the fritted funnel overnight at room temperature for 10 min. Furtherdrying was carried out on high vacuum at 25° C. for 18 h. The finalproduct Linker 1 was obtained as a yellow solid. Yield: 5.786 g, 13.63mmol, 98.7%, HPLC (method 9, 220 nm)>96%. LRMS-ESI (m/z) caled. forC17H₁₉N₄O₇S [M−H]⁻: 423.10. Found: 422.95.

Example 2 Preparation of Keto-Maytansinoids by Direct Esterification ofMaytansinol with a Keto Acid

General method A: Maytansinol (500 mg, 0.88 mmol, 1 eq) and therespective keto acid (3.52 mmol, 4 eq) were dissolved in anhydrous DCM(40 mL) in the presence of activated molecular sieves and cooled down to4-5° C. using an ice bath. To this solution was then added within 10 s asolution of zinc(II) chloride in diethyl ether (2.64 mL, 2.64 mmol, 3.0eq, 1 M solution). The resulting solution was stirred for 40 min at 4-5°C. followed by the addition of N,N′-diisopropylcarbodiimide (0.55 mL,3.52 mmol, 4 eq). The mixture was stirred at 4° C. and then allowed toreach room temperature slowly overnight, immersed in an ice-bath.Conversion was monitored by LC-MS, and after reaching 60%, the reactionmixture was concentrated under reduced pressure at 40° C. to half of thevolume, filtered through a 0.45 μm syringe filter (Macherey-Nagel,Chromafil® PTFE-O-45/25), and the filtrate was evaporated. The finalproduct was purified with a Biotage Isolera One flash purificationSystem, with a pre-packed SNAP ULTRA 50 g cartridge, with Biotage®HP-Sphere™ spherical silica (linear gradient from 100% DCM to 90/10DCM/methanol in 25 CV). The tubes containing the product were combinedand dried for 1 h with a rotary evaporator, and under high vacuum toafford the respective keto maytansinoid.

General method B: Maytansinol (758 mg, 1.34 mmol, 1.0 eq), therespective keto acid (1.47 mmol, 1.1 eq), and DMAP (181 mg, 1.47 mmol,1.1 eq) were dissolved under N₂ atmosphere in anhydrous DCM (30 mL) inthe presence of activated molecular sieves (0.8 g, 4 Å, 325 meshparticle size, Sigma Aldrich). The mixture was cooled down within 10 minto 4° C. using an ice/water bath. A solution of EDC-HCl (283 mg, 1.47mmol, 1.1 eq) in dry DCM (15 mL) was added within 30 min (˜10 mg/min) tothe cooled mixture and the reaction mixture was stirred at 4° C. for 2h. After this time, another portion of the keto acid (1.47 mmol, 1.1 eq)was added, followed by the addition of a solution of EDC-HCl (283 mg,1.47 mmol, 1.1 eq) in dry DCM (10 mL) within 30 min (˜10 mg/min) to thecooled mixture and the reaction mixture was stirred under inertatmosphere at 4° C. for 2 h. After this time, the addition of reagentswas repeated once more, and the reaction mixture was left stirringovernight at 4° C. and then allowed to reach room temperature graduallyduring this time. The mixture was filtered (Macherey-Nagel, Chromafil®PTFE-O-45/25), and the solvent was removed with a rotary evaporator at40° C. to a final volume of approximately 10 mL. The crude was purifiedwith a Biotage Isolera One flash purification System, with a pre-packedSNAP ULTRA 100 g cartridge, with Biotage® HP-Sphere™ spherical silica(linear gradient from 100% DCM to 90/10 DCM/methanol in 25 CV). Thetubes containing the product were combined and the solvent was removedwith a rotary evaporator to obtain a solid. The solid was dried underhigh vacuum to afford the respective keto maytansinoid.

Preparation of Maytansinoid 2

From the reaction of maytansinol with 2-(4-acetyl-2-fluorophenyl)aceticacid using Method A: Maytansinoid 2 was obtained as a yellowish solid.Yield: 47%. Purity by RP-HPLC, 220 nm, 96%. LRMS-ESI (m/z) calcd. for:C38H₄₅ClFN₂O₁₀ [M+H]⁺: 743.22. Found: 743.25. LRMS-ESI (m/z) calcd. for:C38H₄₃ClFN₂O₁₀ [M−H]⁻: 741.22. Found: 741.41.

The structure was confirmed by ¹H NMR and ¹³C NMR: ¹H NMR (400 MHz,CDCl₃) δ 7.73 (dd, J=7.9, 1.6 Hz, 1H; C31-CH), 7.66 (dd, J=10.5, 1.6 Hz,1H; C27-CH), 7.46 (t, J=7.6 Hz, 1H; C32-CH), 6.82 (d, J=1.8 Hz, 1H;C17-CH), 6.59 (d, J=1.8 Hz, 1H; C21-CH), 6.48 (dd, J=15.5, 11.0 Hz, 1H;C12-CH), 6.43 (s, 1H; C9-NH), 6.25 (d, J=10.9 Hz, 1H; C13-CH), 5.63 (dd,J=15.4, 8.8 Hz, 1H; C11-CH), 4.99 (dd, J=11.8, 2.7 Hz, 1H; C3-CH), 4.27(td, J=11.2, 10.4, 1.8 Hz, 1H; C7-CH), 3.97 (s, 3H; C20-OCH₃), 3.90 (d,J=15.7 Hz, 1H; C24-CH₂), 3.76 (d, J=15.5 Hz, 1H; C24-CH₂), 3.54 (d,J=8.8 Hz, 1H; C10-CH), 3.46 (d, J=12.8 Hz, 1H; C15-CH₂), 3.38 (s, 3H;C10-OCH₃), 3.19 (d, J=12.8 Hz, 1H; C15-CH₂), 3.00 (s, 3H; C1-NCH₃), 2.87(d, J=9.7 Hz, 1H; C5-CH), 2.57 (s, 3H; C29-CH₃), 2.51 (dd, J=14.1, 11.9Hz, 1H; C2-CH₂), 2.17 (dd, J=13.9, 2.6 Hz, 1H; C2-CH₂), 1.72 (d, J=13.6Hz, 1H; C8-CH₂), 1.68 (s, 3H; C14-CH₃), 1.50 (m, 1H; C6-CH), 1.28 (d,J=6.3 Hz, 4H; C6-CH₃, C8-CH₂), 0.87 (d, J=1.4 Hz, 3H; C4-CH₃); ¹³C NMR(101 MHz, CDCl₃) δ 196.50 (C28), 168.86 (C23), 168.30 (C1), 160.78 (d,¹J_(C-F)=247.0 Hz; C26), 156.13 (C20), 152.46 (C22), 142.53 (C18),140.30 (C19), 140.24 (C14), 138.49 (d, ³J_(C-F)=6.4 Hz; C30), 132.58(C12), 131.68 (d, ³J_(C-F)=3.6 Hz; C32), 128.32 (C11), 126.38 (d,²J_(C-F)=16.1 Hz; C25), 124.82 (d, ⁴J_(C-F)=3.3 Hz; C31), 124.56 (C13),122.04 (C21), 119.52 (C16), 114.73 (d, ²J_(C-F)=23.2 Hz; C27), 113.13(C17), 88.23 (C10), 81.25 (C9), 77.95 (C3), 74.37 (C7), 66.24 (C5),60.39 (C4), 56.91 (C20-OCH₃), 56.71 (C10-OCH₃), 47.26 (C15), 38.37 (C6),36.01 (C8), 35.46 (C1-NCH₃), 33.86 (C24), 32.76 (C2), 26.78 (C29), 15.88(C14-CH₃), 14.60 (C6-CH₃), 12.35 (C4-CH₃).

Preparation of Maytansinoid 3

From the reaction of maytansinol with 2-(3-acetylphenoxy)acetic acidusing Method B: Maytansinoid 3 was obtained as a yellowish solid. Yield:49%. Purity by RP-HPLC, 220 nm, 98%. LRMS-ESI (m/z) calcd. for:C₃₈H₄₆ClN₂O₁₁ [M+H]⁺: 741.23. Found: 741.23.

The structure was confirmed by ¹H NMR and ¹³C NMR: ¹H NMR (400 MHz,CDCl₃) δ 7.58 (dt, J=7.8, 1.1 Hz, 1H; C30-CH), 7.55 (s, 1H; C26-CH),7.44 (d, J=7.9 Hz, 1H; C31-CH), 7.12 (dd, J=8.3, 2.8 Hz, 1H; C32-CH),6.79 (d, J=1.8 Hz, 1H; C17-CH), 6.65 (d, J=1.8 Hz, 1H; C21-CH), 6.46(dd, J=15.4, 11.0 Hz, 1H; C12-CH), 6.37 (s, 1H; C9-NH), 6.26 (d, J=11.0Hz, 1H; C13-CH), 5.62 (dd, J=15.4, 8.9 Hz, 1H; C11-CH), 5.08 (dd,J=11.9, 2.7 Hz, 1H; C3-CH), 4.90 (d, J=15.9 Hz, 1H; C24-CH₂), 4.66 (d,J=15.9 Hz, 1H; C24-CH₂), 4.28 (t, J=10.6 Hz, 1H; C7-CH), 3.96 (s, 3H;C20-OCH₃), 3.68 (s, 1H; C9-OH), 3.53 (d, J=8.9 Hz, 1H; C10-CH), 3.47 (d,J=12.9 Hz, 1H; C15-CH₂), 3.36 (s, 3H; C10-OCH₃), 3.15 (d, J=12.8 Hz, 1H;C15-CH₂), 2.92 (d, J=9.7 Hz, 1H; C5-CH), 2.87 (s, 3H; C1-NCH₃), 2.59 (s,3H; C29-CH₃), 2.55 (d, J=11.9 Hz, 1H; C2-CH₂), 2.22-2.14 (m, 1H;C2-CH₂), 1.71 (d, J=1.8 Hz, 1H; C8—CH₂), 1.67 (s, 3H; C14-CH₃),1.55-1.45 (in, 1H; C6-CH), 1.28 (d, J=6.4 Hz, 3H; C6-CH₃), 1.28-1.25 (m,1H; C8-CH₂), 0.86 (s, 3H; C4-CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 197.99(C28), 168.09 (C1), 168.07 (C23), 158.27 (C25), 156.09 (C20), 152.36(C22), 142.46 (C18), 140.38 (C19), 139.83 (C14), 138.84 (C27), 132.85(C12), 130.48 (C31), 128.22 (C11), 124.95 (C13), 122.41 (C30), 122.12(C21), 119.67 (C32), 119.25 (C16), 113.73 (C26), 113.02 (C17), 88.18(C10), 81.16 (C9), 78.13 (C3), 74.18 (C7), 66.37 (C24), 66.26 (C5),60.26 (C4), 56.87 (C20-OCH₃), 56.68 (C10-OCH₃), 47.05 (C15), 38.41 (C6),36.34 (C8), 35.31 (C1-NCH₃), 32.67 (C2), 26.88 (C29), 15.86 (C14-CH₃),14.58 (C6-CH₃), 12.50 (C4-CH₃).

TABLE 1 Maytansinoids synthesized using Method A or B Com AA poundStructure Spacer Method Yield  4

— A 53%  5

— A 66%  6

— A 27%  7

— A 25%  8

— A 27%  9

— A 13% 10

— A 33% 11

— A 72% 12

— A 72% 13

— A 47% 14

— A 45% 15

— B  4% 16

— B  6% 17

— B 11% 18

— B 49% 19

— B 13% 20

Pro A  5% 21

Gly A  6% 22

N-Et-Gly A  9% 23

Pro A 21%

Example 3

Three-step synthesis of keto-maytansinoids via esterification with anFmoc-protected amino acid, cleavage of Fmoc group and condensation witha keto acid.

General method C—Step 1: reaction of maytansinol with an Fmoc-protectedamino acid. Maytansinol (565 mg, 1.00 mmol, 1.0 eq), the Fmoc-protectedamino acid (3.00 mmol, 3.0 eq), DMAP (982 mg, 8.00 mmol, 8.0 eq), andscandium (III) trifluoromethanesulfonate (541 mg, 1.1 mmol, 1.1 eq) weredissolved under N₂ atmosphere in anhydrous DCM (10 mL) at the presenceof activated molecular sieves (1 g, 4 Å, 325 mesh particle size, SigmaAldrich). The mixture was cooled to 4° C. using an ice/water bath andleft stirring for 30 min to reach that temperature. After this time DIC(2.47 mL, 16.0 mmol, 16.0 eq) was added within 10 min (˜0.25 mL/min) andthe reaction mixture was stirred at 4° C. for 2 h and allowed to reachroom temperature gradually during this time. The mixture was filtered bygravity. The filtrate was diluted with DCM (50 mL) and was then washedwith sodium phosphate buffer (50 mL×3, pH 7.5) and brine (50 mL). Theorganic layer was then dried over anhydrous sodium sulfate, filtered bygravity, and the solvent was removed with a rotary evaporator at 40° C.to a final volume of approximately 10 mL. The crude was purified on aBiotage Isolera One flash purification System, with a pre-packed SNAPULTRA 50 g cartridge, with Biotage® HP-Sphere™ spherical silica (lineargradient from 100% DCM to 90/10 DCM/methanol in 25 CV). The solvent wasremoved with a rotatory evaporator at 40° C. for 2 h to obtain therespective product as a yellowish to yellow solid.

General method C—Step 2: deprotection of the Fmoc group. TheFmoc-protected intermediate (0.53 mmol, 1.0 eq) was dissolved in DCM (5mL), and to this solution was added tris-(2-aminoethyl)amine (0.320 mL,2.13 mmol, 4.0 eq) within 10 s. The reaction mixture was left stirringat room temperature for 1 h. The white precipitate formed was filteredoff over a Celite pad, washed with DCM (20 mL), and the yellow filtratewas evaporated to dryness with a rotary evaporator at 40° C. for 2 h,and further dried under high vacuum for 4 h to afford the free aminewhich was immediately used in the next step without furtherpurification.

General method C—Step 3: reaction of the amine-maytansinoid with a ketoacid. The free amine intermediate (77.0 μmol, 1.0 eq), the keto acid(150 μmol, 2.0 eq), HATU (35 mg, 94.0 μmol, 1.2 eq), HOAt (13 mg, 94.0μmol, 1.2 eq), and N-methylmorpholine (17 μL, 150 μmol, 2.0 eq) weredissolved under N₂ atmosphere in anhydrous DMF (1 mL). The mixture wasleft stirring at room temperature overnight. The reaction mixture wasdiluted with DCM (5 mL) and was washed with a saturated solution ofammonium chloride (5 mL×5) and brine (5 mL). The organic phase was thendried over anhydrous sodium sulfate, filtered by gravity and the solventwas removed with a rotary evaporator at 40° C. The crude was purified ona Biotage Isolera One flash purification System with a pre-packed SNAPULTRA C-18 12 g cartridge, Biotage® HP-Sphere™ C18, 25 μm sphericalsilica (linear gradient system from 80/20 water/MeCN to 100% MeCN in 20CV). The product-containing fractions were combined, frozen in liquidnitrogen, and lyophilized for 24 h to afford the respective ketomaytansinoid.

TABLE 2 Maytansinoids synthesized using Method C (steps 1-3) Com- AAYield Yield Yield pound Structure Spacer step 1 step 2 step 3 44

N-Me-Ala 53% 87% 30% 24

N-Me-Ala 53% 87% 16% 25

Sar 47% 98% 23% 26

Sar 47% 98%  8% 27

β-Ala 58% 95% 12%

Example 4 Preparation of Maytansinoid 28

Maytansinol (56.5 mg, 0.10 mmol, 1.0 eq) was dissolved in dry DCM (10mL), a solution of zinc(II) chloride in diethyl ether (0.3 mL, 0.30mmol, 3.0 eq, 1 M solution) was added at room temperature under N₂atmosphere and left stirring for 10 min. 4-Acetylphenylisocyanate (48.3mg, 0.30 mmol, 3.0 eq) was added, within 10 s, and the resultingsolution was stirred at room temperature for 5 h. After this time,completion of the reaction was confirmed by HPLC analysis (PDA 220 nm).The volatiles were removed with a rotatory evaporator at 40° C. Thecrude was purified by NP chromatography using a Biotage Isolera Oneflash purification System with a pre-packed SNAP ULTRA 10 g cartridgecontaining Biotage® HP-Sphere™ spherical silica (linear gradient systemfrom 100% DCM to 90/10 DCM/methanol in 13 CV) followed by RPchromatography with a pre-packed SNAP ULTRA C-18 12 g cartridge Biotage®HP-Sphere™ C18, 25 μm spherical silica (linear gradient from 80/20water/MeCN to 100% MeCN in 30 CV). The product-containing fractions werecombined, frozen in liquid nitrogen, and lyophilized for 24 h to affordcompound 28 as a white solid. Yield: 20 mg (28%). Purity by RP-HPLC (220nm)≥95%. LRMS-ESI (m/z) calcd. for: C₃₇H₄₅ClN₃O₁₀ [M+H]⁺: 726.27. Found:726.25. LRMS-ESI (m/z) calcd. for: C₃₇H₄₃ClN₃O₁₀ [M−H]⁻: 724.27. Found:724.43.

Example 5 Preparation of Maytansinoid 29

Synthesis of May-ONp: To a solution of maytansinol (88 mg, 155 μmol, 1.0eq) in DCM (8 mL) was added pyridine (25 μL, 310 μmol, 2.0 eq) at roomtemperature. The resulting clear solution was stirred for 15 min beforecooling down to 4° C. and adding p-nitrophenyl chloroformate (219 mg,1.08 mmol, 7.0 eq) in DCM (4 mL) after which a white precipitate wasformed immediately. The reaction mixture was stirred for 24 h at roomtemperature. The crude material was concentrated using a rotaryevaporator at 40° C. for 1 h and was purified with a Biotage Isolera Oneflash purification System using a pre-packed SNAP ULTRA 25 g cartridgewith Biotage® HP-Sphere™ spherical silica (linear gradient from 100%ethyl acetate to 40/60 ethyl acetate/DCM in 50 CV). Theproduct-containing fractions were dried with the rotary evaporator at40° C. for 30 min and under high vacuum for another 30 min to afford theintermediate May-ONp as a white solid. Yield: 102 mg (90%). LRMS-ESI (m% z) calcd. for C₃₅H₄₁ClN₃O₁₂ [M+H]⁺: 730.23. Found: 730.01.

Synthesis of maytansinoid 29: To a solution of compound May-ONp (3.7 mg,5.00 μmol, 1.0 eq) in DCM (1 mL) was added a solution of4-(aminomethyl)acetophenone (1.1 mg, 7.50 μmol, 1.5 eq) in DCM/DMF(2:0.1 v/v) at room temperature and was stirred for 5 min before addingtriethylamine (2 μL, 10.0 μmol, 2.0 eq). The reaction mixture wasstirred at room temperature for 2 h and at 60° C. overnight. The crudematerial was concentrated at 40° C. for 1 h and was purified with aBiotage Isolera One flash purification System using a pre-packed SNAPULTRA 10 g cartridge with Biotage® HP-Sphere™ spherical silica (lineargradient from 100% chloroform until 90/10 chloroform/methanol in 20 CV).The product-containing fractions were dried with the rotary evaporatorat 40° C. for 30 min and under high vacuum for another 30 min to affordcompound 29 as a white solid. Yield: 1 mg (27%). Purity by RP-HPLC (220nm)≥95%. LRMS-ESI (m/z) caled. for C₃₈H₄₆ClN₃O₁₀ [M+H]⁻: 739.28. Found:739.96.

Example 6 Preparation of Albumin-Binding Maytansinoids

General method D for the synthesis of albumin-binding maytansinoids froma keto-maytansinoid and a maleimido-hydrazide linker: To a stirredsolution of the keto-maytansinoid (1.0 eq), in dry solvent (suitablesolvents are DCM, DMSO, dioxane, 2-methyltetrahydrofuran) at roomtemperature under N₂ atmosphere were added molecular sieves (from 1:1 to5:1 w/w) and a catalyst (TFA, p-toluenesulfonic acid, amberlyst-H form,amberlite-Na form) followed by a solution of the hydrazide linker (from1.0-5.0 eq) in dry DMSO. The reaction mixture was stirred at roomtemperature and conversion was confirmed using HPLC analysis (PDA 220nm) (>90% conversion). The reaction mixture was filtered, concentratedwith a rotary evaporator at 30° C. and purified by chromatography with aBiotage Isolera One flash purification System using a pre-packed SNAPULTRA Biotage® HP-Sphere™ spherical silica (linear gradient from 100%DCM to 90/10 DCM/methanol), dried with the rotary evaporator at 30° C.for 30 min, and under high vacuum for another 30 min to afford the freeacid. The product was re-dissolved in suitable solvent (methanol,acetone, 2-methyltetrahydrofuran, or other organic polar solvents) andthen was neutralized using salts solutions (sodium salts, potassiumsalts, triethylammonium salts) until a pH range 5.5-7.5 was reached. Thesolution was frozen in liquid nitrogen, and lyophilized for 24 h toafford the compound as a salt. The content of the counterion wasdetermined by IC. The sodium ion content ranged from 0.2-1.0 eq (ca.0.2-0.9%) and the triethylammonium ion content from 0.8-2.0 eq (ca.6-16%).

Preparation of Albumin-Binding Maytansinoid 30

From the reaction of maytansinoid 2 with linker 1: To a stirred solutionof maytansinoid 2 (170 mg, 0.23 mmol, 1.0 eq), molecular sieves (0.2 g,powder, activated, 4 Å, 325 mesh particle size), and Amberlyst®-H (20mg, macroporous, 30-60 mesh) in dry DCM (2 mL) at room temperature underN₂ atmosphere was added a solution of the linker 1 (51 mg, 0.12 mmol,2.0 eq) in dry DMSO (0.6 mL). The reaction mixture was stirred at roomtemperature, and after 3 h HPLC analysis (PDA 220 nm) confirmedcompletion of the reaction (>98% conversion). The reaction mixture wasconcentrated with a rotary evaporator at 30° C., filtered over a 0.45 μmsyringe filter (Macherey-Nagel, Chromafil® PTFE-O-45/25). The filtratewas diluted with dry DCM (8 mL) and was purified with a Biotage IsoleraOne flash purification System using a pre-packed SNAP ULTRA 10 gcartridge with Biotage® HP-Sphere™ spherical silica (linear gradientfrom 100% DCM to 90/10 DCM/MeOH in 22 CV). The combinedproduct-containing fractions were dried with the rotary evaporator at30° C. for 30 min and under high vacuum for another 30 min to afford thefree acid form of 30 as a white-yellow solid. The solid was dissolved inMeOH/acetone (50:50 v/v, ca. 4 mL in total) and then it was neutralizedwith a 5 mM solution of NaHCO₃ in Millipore water until pH 6.8-7.1 (pHmeasured using pH meter). The solution was frozen in liquid nitrogen,and lyophilized for 24 h to afford maytansinoid-prodrug 30 as awhite-yellow foam. Yield: 161 mg (60%). LRMS-ESI (m/z) calcd. for:C₅₅H₆₁ClFN₆O₁₆S [M−H]⁻: 1147.36. Found: 1147.84. The content of Na⁺ranged from 0.2-0.8%.

Preparation of Albumin-Binding Maytansinoid 31

Prom the reaction of maytansinoid 3 with linker 1: To a stirred solutionof maytansinoid 3 (186 mg, 0.25 mmol, 1.0 eq), molecular sieves (0.4 g,powder, activated, 4 Å, 325 mesh particle size, Sigma Aldrich), andAmberlite® (IR120 Na form, 484 mg, 2.1 mmol/mL, 4.0 eq) in drydichloromethane (4 mL) at room temperature under N₂ atmosphere was addeda solution of linker 1 (329 mg, 0.77 mmol, 2.5 eq) in dry DMSO (4 mL).The reaction mixture was stirred at room temperature, and after 6 h HPLCanalysis (PDA 220 nm) confirmed completion of the reaction (>98%conversion). The reaction mixture was concentrated with a rotaryevaporator at 30° C., filtered over a 0.45 μm syringe filter(Macherey-Nagel, Chromafil® PTFE-0-45/25). The filtrate was neutralizedwith sodium hydroxide (318 μL of a IM NaOH solution, 1.3 eq). Theneutralized mixture was added dropwise to a 50 mL Falcon tube containinga cooled mixture (4° C. ice bath) of methyl t-butyl ether (27 mL) andisopropyl alcohol (14 mL) and centrifuge at 4° C. for 5 min. Thesupermatant was decanted, and the precipitate was re-suspended indichloromethane (10 mL) and purified with a Biotage Isolera One flashpurification System using a pre-packed SNAP ULTRA 10 g cartridge, withBiotage® HTP-Sphere™ spherical silica (linear gradient from 100% DCMuntil 90/10 DCM/MeOH in 22 CV). The combined product-containingfractions were dried with the rotatory evaporator at 30° C. for 30 minand under high vacuum for another 10 h to afford 31 as a white-yellowsolid. Yield: 156 mg (52%). Purity by RP-HPLC (220 nm)≥95%. LRMS-ESI(m/z) calcd. for: C₅₅H₆₂ClN₆O₁₇S [M−H]⁻: 1145.36. Found: 1145.87. Thecontent of Na has a window of 0.2-0.8%.

TABLE 3 Yield Com- Drug (counter pound Structure (Cmpd) ion) 45

 4 54% (−) 32

 4 10% (Na⁺) 33

 4 33% (Na⁺) 34

 4 49% 35

 2 33% (Na⁺) 36

18 17% (Et₃NH⁺) 37

11 12% (Et₃NH⁺) 38

13 24% (Et₃NH⁺) 39

12 24% (Et₃NH⁺) 40

10 12% (Et₃NH⁺) 41

 9 7% (Na⁺) 42

 3 73% (Na⁺) 43

18 11% (Na⁺)

Example 7 Stability and Release Kinetics of Maytansinoid-HSA Conjugatesin Buffer Solution at pH 4.0 and pH 7.4

For preparing HSA conjugates of albumin-binding maytansinoids, HSA (200μM: 361.8 μL, 1078 μM free Cys34; 100 μM: 180.9 μL, 1078 μM free Cys34)was diluted with PBS buffer (4 mM sodium phosphate, 150 mM NaCl, pH 7.4)(200 μM: 678.2 μL; 100 μM: 859.1 μL) and DMSO (200 μM: 130.0 μL; 100 μM:195.0 μL) and incubated in a heating block at 37° C. for 30 minutes. Thealbumin-binding maytansinoid was added as a 2 mM stock solution in DMSO(200 μM: 130.0 μL; 100 μM: 65.0 μL) to the preincubated HSA sample toproduce a 200 μM or 100 μM solution of maytansinoid-prodrug and 300 μMor 150 μM of free Cys34. The mixture having a pH of 7.4 was allowed toreact for 10 min at 37° C. and was then analyzed hourly for 24 hours byRP-HPLC (Phenomenex Aeris WP XB-C18, 3.6 μm, 250×4.6 mm).

For studying the release kinetics at pH 4.0 the mixture was acidifiedwith a mixture of 50 mM sodium acetate buffer pH 3.0 (200 μM: 119.3 μL;100 μM: 125.2 μL) and 1 M HCl (200 μM: 12.7 μL; 100 μM: 6.8 μL) to reachpH 4.0. The mixture was subsequently analyzed hourly for 24 hours byRP-HPLC (Phenomenex Aeris WP XB-C18, 3.6 μm, 250×4.6 mm).

The following RP-HPLC conditions were used: Phenomenex Aeris WP XB-C183.6 μm, 250×4.6 mm; eluent A (100% 20 mM Tris buffer pH 8.0) and eluentB (90:10, MeCN: water) eluting with a gradient of eluent B (25% 0-0.5min, 25-35% 0.5-2.5 min, 35-85% 2.5-16 min, 85-95% 16-17 min, 95% 17-20min, 95-25% 20-25 min, 25% 25-30 min, flow rate 1.0 mL/min). Column oventemperature 37° C.; autosampler temperature 37° C.; 20 μL of injectionvolume.

To quantify the percentage of free drug released, standard curves of thefree maytansinoids were prepared at different concentrations (200 μM,100 μM, 50 μM, 25 μM and 12.5 μM). The area under the curve (AUC) wasdetermined at 250 nm.

TABLE 4 Release kinetics of maytansinoid-HSA conjugates at pH 4.0 and pH7.4 free maytansinoid Maytansinoid rel. t_(1/2) at pH 4.1 after 24 h atpH 7.4 36 4.5 h* 4.7%* 41 4.0 h* 4.6%* 31 4.5 h* 4.0%* 6.0 h**  3.7%**33 7.0 h* 6.5%* 32 4.0 h* 9.5%* 37 5.5 h* 3.9%* 30 3.5 h**  9.6%** 403.5 h* 8.9%* 39 5.5 h* 9.6%* 38 8.5 h* 4.9%* 42 45.4 h**  4.8%***measured at 200 μM **measured at 100 μM

Example 8

Stability of maytansinoids and maytansinoid-HSA conjugates in CD1 mouseand humanplasma: For studying the stability of the maytansinoids aswells as the maytansinoid-HSA conjugates in CD1 and human plasma thecompounds were incubated at 37° C. for 24 hours. Remaining freemaytansinoid or release of the respective maytansinoid was quantified byLC-MS/MS (or HPLC) at certain time points.

LC-MS quantification procedure: Pooled CD1 mouse or human plasma(K₂EDTA, Innovative research) was centrifuged (1 min at 12,044 g). Thesupernatant was first filtered through a filter needle (5 μm, sterile,Becton Dickinson) and subsequently through a cellulose acetate membrane(0.45 μm, sterile, Carl Roth). The filtered plasma (1710 μL) waspreincubated at 37° C. for 30 min in a heating block. The freemaytansinoid or albumin-binding maytansinoid as a 300 μM stock solutionin DMSO (190 μL) was added to the preincubated plasma sample to producea 30 μM solution of the respective maytansinoid or maytansinoid-HSAconjugate. The mixture was allowed to react for 10 min at 37° C. andthen at each time point (0, 1, 2, 3, 4, 5, 21, and 24 hours) threesamples (70 μL) were taken into a 96-well plate, sealed with a plasticmat, immediately frozen with liquid nitrogen and stored at −20° C. Atthe end of the experiment, the 96-well plate was thawed at roomtemperature. The samples (30 μL) were then directly transferred via amultichannel pipette into a 96-well Impact™ protein precipitation plate(Strata*) which was once washed with MeCN (150 μL) and then loaded withthe internal standard (120 μL, MeCN containing 5 μg/mL maytansine). TheImpact™ precipitation plate was sealed with a silicone mat and shakenfor 2 min 420 rpm. The Impact™ precipitation plate was then placed ontoa 96-well sample manifold and the filtrate was collected into another96-well plate by applying mild vacuum. The 96-well plate was sealed witha silicone mat and kept at room temperature until LC-MS/MSquantification. The filtrate was injected into the LC-MS forquantification using the MRM mode.

LC-MS method: Phenomenex Luna® Omega Polar C18, 1.6 μm, 100 Å, 50×2.1mm, column; eluent A (0.1% formic acid in water) and eluent B (0.1%formic acid in MeCN) eluting with a gradient of eluent B (20% 0-0.5 min,flow 0.4 mL/min; 20-60% 0.5-9.0 min, 0.4 mL/min; 60-100% 9.0-9.5 min,flow 0.4 mL/min; 100-20% 9.5-10.5 min, flow 0.4 mL/min; 20% 10.5-12 min,0.6 mL/min). Column oven temperature 25° C.; autosampler temperatureroom temperature; 10 μL of injection volume.

The area under the curve (AUC) for each free drug at time point 0 wasset as 100% value for the respective prodrug. The AUC for each samplewas normalized based on the AUC of maytansine.

HPLC quantification procedure: Samples were prepared as described beforeusing a 2 mM stock solution in DMSO of the free drug as well as theprodrug. After incubation at 37° C. the samples were directly injectedinto HPLC every hour for a period of 24 hours. The area under the curve(AUC) for each free maytansinoid (200 μM in 10% DMSO in PBS buffer) wasused as 100% value for the respective albumin-binding maytansinoid.

HPLC method: Phenomenex Aeris WP XB-C18, 3.6 μm, 250×4.6 mm, column;eluent A (20 mM Tris buffer pH 8.0) and eluent B (90:10, MeCN: water)eluting with a gradient of eluent B (25% 0-0.5 min, 25-35% 0.5-2.5 min,35-85% 2.5-16 min, 85-95% 16-17 min, 95% 17-20 min, 95-25% 20-25 min,25% 25-30 min, flow rate=1.0 mL/min). Column oven temperature 37° C.;autosampler temperature 37° C.; 20 μL of injection volume.

The relative release of the free maytansinoid as well as the conversioninto maytansinol are listed below in Table 5.

TABLE 5 Relative release of the free maytansinoid and/or maytansinol inCD1 mouse and human plasma for the maytansinoid-HSA conjugates Albumin-binding % of free maytansinoid % of maytansinol maytansinoid releasedafter 24 h conversion after 24 h (Cmpd) CD1 mouse human CD1 mouse human31 3.0 6.8 2.9 2.1 45 6.5 na nd na  34* 4.3 na nd na 33 8.7 10.3  nd nd32 7.0 7.8 nd 0.3 30 4.1 6.2 nd nd 42 2.7 7.4 2.1 1.8 35 na 9.0 na nd*data were obtained by HPLC

The amount of remaining free maytansinoid as well as the conversion intomaytansinol are listed below in Table 6.

TABLE 6 Remaining amount of free maytansinoid and maytansinol after 24 hin CD1 mouse and human albumin % of free maytansinoid % of maytansinolMaytansinoid remaining after 24 h conversion after 24 h (Cmpd) CD1 mousehuman CD1 mouse human 4 65.2 75.6  0.5 nd 2 71.8 74.8 nd nd 3 40.8 69.619.1 4.5

The stability of different linkers with (maytansinoid 4) in CD1 murineplasma is depicted in FIG. 1 .

Example 9

In vitro binding kinetics of albumin-binding maytansinoids to albumin inpooled human whole blood and plasma: To study the binding kinetics ofthe albumin-binding maytansinoids in pooled human plasma and pooledhuman whole blood, the compounds were incubated together with pooledhuman plasma at 37° C. and samples were taken at specified time points.Remaining albumin-binding maytansinoids were quantitated by HPLC.

HPLC quantification procedure: To study the binding kinetics in humanwhole blood, the blood (K₂EDTA, 36 donors, Zen-Bio; 900 μL) waspreincubated at 37° C. for 30 min in a heating block. To study thebinding kinetics in pooled human plasma, the pooled whole blood wascentrifuged (10 min, 1811 g), the supernatant plasma was collected andthen preincubated at 37° C. for 30 min.

The albumin-binding maytansinoid was added to the preincubated wholeblood/plasma sample as a 10-fold dilution in PBS of a 1.2 mM stocksolution in 2.5% propylene glycol in 10 mM sodium phosphate buffer and1.48 mM citrate (100 μL) to produce a 12.0 μM solution of the respectivealbumin-binding maytansinoid. Samples (190 μL) were taken after 15 s, 2min, 4 min, 8 min and 15 min. The samples were directly added to 760 μLMeCN containing maytansine (5 μg/mL) as an internal standard andvortexed for 1 min. After centrifugation (1 min at 12,044 g) thesupernatant (850 μL) was concentrated under high vacuum. The residue wastaken up in DMSO/water (1:1 v/v; 95 μL) and then analyzed by RP-HPLC.Percentage of binding was determined by comparison of the area under thecurve (AUC) at 220 nm of the albumin-binding maytansinoid relative to acontrol sample (100% value) in PBS buffer. All experiments wereperformed in triplicates.

HPLC method: Phenomenex Kinetex Polar C18, 2.6 μm, 100 Å, 150×4.6 mm;eluent A (95:5 5 mM ammonium acetate: MeCN) and eluent B (5:95 5 mMammonium acetate: MeCN) eluting with a gradient of eluent B (30% 0-0.5min, 30-95% 0.5-9.0 min, 95% 11.0 min, 95-30% 11.0-14.5 min, 30% 15.0min, flow rate=1.0 mL/min). Column oven temperature ambient temperature;autosampler temperature 4° C.; 50 μL of injection volume.

The relative amount of bound albumin-binding maytansinoid at 0 and 8 minare listed in the table below:

TABLE 7 Amount of bound albumin-binding maytansinoid after 15 s and 8min in pooled human whole blood and plasma % bound Albumin-bindingplasma whole blood maytansinoid 15 s 8 min 15 s 8 min 31 92.0 ± 7.5 98.6± 0.1 99.2 ± 0.4 99.3 ± 0.2 30 96.4 ± 3.6 98.5 ± 0.6 98.3 ± 0.5 98.5 ±0.6

Example 10 In Vitro Cytotoxicity of Maytansine, DM1, DM1-SMe,Maytansinol, and the Maytansinoids in Different Cell Lines Using theCellTiter-Blue® Cell Viability Assay

The study tested the in vitro efficacy of all compounds using Promega'sCellTiter-Blue® Cell Viability Assay. The cancer cell lines that weretested are: LXFL 529 (large cell lung cancer), RKO (colon cancer),SW-620 (colon cancer), CAL-27 (head & neck cancer), LXFL 1674L (largecell lung cancer), MDA-MB 468 (mammary cancer), SK-OV-3 (ovariancancer), PAXF 1657 (pancreatic cancer), MCF7 (mammary cancer), COLO 205(colon cancer), MDA-MB 231 (mammary cancer), BT-474 (mammary cancer),and Hep G2 (liver cancer).

Cells are harvested from exponential phase cultures, counted and platedin 96 well flat-bottom microtiter plates at a cell density depending onthe cell line's growth rate (4,000 and 60,000 cells). After a 24 hoursrecovery period to allow the cells to resume exponential growth, 10 μLof culture medium (four control wells/plate) or of culture medium withthe test compound are added.

Compounds are applied in half-log dilution steps at 10 concentrations induplicate and cells are treated continuously for 96 h. After treatmentand incubation of the cells, 20 μL/well CellTiter-Blue® reagent isadded. After incubation of up to 4 hours, fluorescence (FU) is measuredby using the EnSpire multilabel reader (excitation λ=531 nm, emissionk=615 nm). Sigmoidal concentration response curves are fitted to thedata points (T/C values) obtained for each cell line using 4 parameternon-linear curve fit (Oncotest Warehouse Software). IC₅₀ values arereported as relative IC₅₀ values, being the concentration of testcompound that give a response (inhibition of colony formation/viability)half way between the top and bottom plateau of the sigmoidalconcentration response curve (inflection point of the curve), or asabsolute IC₅₀ values, being the concentration of test compound at theintersection of the concentration-response curves with T/C=50%. Forcalculation of mean IC₅₀ values the geometric mean is used. Results arepresented as mean graph plots or heat maps (individual IC₅₀ valuesrelative to the geometric mean IC₅₀ value) over all cell lines astested. See Table 8 and FIG. 2 . FIG. 2 shows the heat map of IC₅₀ forall tested compounds.

TABLE 8 Geomean IC₅₀ [nM] n Cell Lines DM1 3.22 6 DM1-SMe 1.30 6 18 0.1312 24 9.04 7 44 7.21 6 4 0.16 13 2 0.30 11 5 0.90 6 6 2.14 6 28 10.6 620 0.94 6 7 0.89 6 23 26.80 10 21 1.67 6 22 25.00 3 19 1.12 6 9 0.19 717 0.45 6 15 0.18 7 16 0.32 7 14 1.54 6 3 0.10 6 8 0.10 6 48 59.90 6Maytansine 0.22 12 Maytansinol 86.00 6 27 11.70 6 46 67.80 6 11 0.21 825 8.47 7 10 0.30 7 47 10.70 7 26 18.50 7 12 0.76 6 13 0.88 6

Example 11 General Procedure for the Evaluation of Maytansine and theAlbumin-Binding Maytansinoid Compounds in a Patient-Derived TumorXenograft Model

Female immunodeficient NMRI nude mice, from Charles River, receivedunilateral tumor implants subcutaneously in the left flank while underisoflurane anesthesia with human cancer tumors until tumors werepalpable and had reached a volume of 80-200 mm³ (unless otherwisestated).

Animals were kept in cages, the temperature inside the cages maintainedat 25±1° C. with a relative humidity of 45-65% and an air change rate inthe cage of 60-fold per hour. They were kept under a 14 h light: 10 hdark, artificial light cycle. The animals were fed with autoclavedTeklad Global 19% Protein Extruded Diet (T.2019S.12) from Envigo RMSSARL and had access to sterile filtered and acidified (pH 2.5) tapwater, which was changed twice weekly. Feed and water were provided adlibitum. Prior to therapy, the animals were randomized (7 mice pergroup, unless otherwise stated) considering a comparable median and meanof group tumor volume. Animals were routinely monitored twice daily onworking days and daily on Saturdays and Sundays. Starting on day 0,animals were weighed twice a week. Relative body weights (RBW) ofindividual animals were calculated by dividing the individual absolutebody weight on day X (BWx) by the individual body weight on the day ofrandomization multiplied by 100%. The tumor volume was determined by atwo-dimensional measurement with calipers on the day of randomization(day 0) and then twice weekly. Tumor volumes were calculated accordingto the following equation: Tumor Vol [mm³]=1 [mm]×w² [mm²]×0.5, where“l” is the length and “w” is width of the tumor. The relative volume ofan individual tumor on day X (RTVx) was calculated by dividing theabsolute individual tumor volume [mm³] of the respective tumor on day X(Tx) by the absolute individual tumor volume of the same tumor on theday of randomization multiplied by 100%. Schedules were applied to theextent that animal welfare policies allow. Termination of individualmice was carried out at tumor volume >2000 mm³ (unilateral). All testcompounds were administered on day 1, 8, 15, and 22 and were supplied aslyophilized solids containing sucrose. On the day of treatment, thelyophilized samples were dissolved in 10 mM sodium phosphate buffer pH6.8, containing 20% propylene glycol and injected intravenously (100μL/20-g mouse) together with vehicle (10 mM sodium phosphate buffer, 20%propylene glycol, and 5% sucrose pH 6.8).

Example 12 General Procedure for the Evaluation of Maytansine and theAlbumin-Binding Maytansinoid Compounds in a Cancer Cell-Line-DerivedXenograft Model

Female immunodeficient NMRI nude mice, from Janvier, France, received5×10⁶ cultured cancer cells in buffer/Matrigel (1:1) transplantsubcutaneously (unless otherwise stated) until tumors were palpable andhad reached a volume of 80-200 mm³ (unless otherwise stated). Animalswere kept in cages, the temperature inside the cages maintained at 22±1°C. with a relative humidity of 50±10% and an air change rate in the cageof 60-fold per hour. They were kept under a 12 h light: 12 h dark,artificial light cycle. The animals were fed with autoclaved Ssniff NM,from Ssniff® and had access to sterile filtered and acidified (pH 4.0)tap water, which was changed twice weekly. Feed and water were providedad libitum. Prior to therapy, the animals were randomized (7 mice pergroup, unless otherwise stated) considering a comparable median and meanof group tumor volume. Animals were routinely monitored twice daily onworking days and daily on Saturdays and Sundays. Starting on day 0,individual body weights of mice were determined two or three times perweek and mean body weight per group was related to the initial value inpercent to calculate the body weight change (BWC). The tumor volume wasdetermined by a two-dimensional measurement with calipers on the day ofrandomization (day 0) and then twice or three times per week. Tumorvolumes were calculated according to the following equation:

Tumor Vol [mm³]=1 [mm]× w² [mm²]×0.5, where “1” is the length and “w” iswidth of the tumor. The relative volume of an individual tumor on day X(RTV_(x)) was calculated by dividing the absolute individual tumorvolume [mm³] of the respective tumor on day X (Tx) by the absoluteindividual tumor volume of the same tumor on the day of randomizationmultiplied by 100%. Schedules were applied to the extent that animalwelfare policies allow. Termination of individual mice was carried outat tumor volume>1500 mm³ (unilateral) or ulceration was observed. Alltest compounds were administered on day 1, 8, 15, and 22 and weresupplied as lyophilized solids containing sucrose. On the day oftreatment, the lyophilized samples were dissolved in 10 mM sodiumphosphate buffer pH 6.8, containing 20% propylene glycol and injectedintravenously (100 μL/20-g mouse) together with vehicle (10 mM sodiumphosphate buffer, 20% propylene glycol, and 5% sucrose—pH 6.8).

Example 13

Evaluation of maytansine and the albumin-binding maytansinoids 30, 42,31 and 35 in the human PDX renal cell cancer model RXF 631

The evaluation of maytansine and the albumin-binding compounds 30, 42,31 and 35 in the renal cancer cell RXF 631 model was carried outaccording to the general procedure for a patient-derived xenograftmodel. Treatment was initiated after tumors reaching an average size of100 mm³. FIG. 3 shows tumor growth curves of the control group,maytansine group, and the groups treated with compounds 30, 42, 31 and35. FIG. 4 shows curves of body weight change in the control group,maytansine group, and the groups treated with compounds 30, 42, 31 and35.

Example 14 Evaluation of Maytansine and the Albumin-BindingMaytansinoids 32, 30, and 31 in the Human PDX Squamous Cell Lung CancerModel LXFE 937

The evaluation of maytansine and the albumin-binding compounds 32, 30,and 31 in the squamous cell lung cancer LXFE 937 model was carried outaccording to the general procedure for a patient-derived xenograftmodel. Treatment was initiated after tumors reaching an average size of117 mm³. FIG. 5 shows tumor growth curves of the control group,maytansine group, and the groups treated with compounds 32, 30, and 31.FIG. 6 shows curves of body weight change in the control group,maytansine group, and the groups treated with compounds 32, 30, and 31.

Example 15 Evaluation of Maytansine and the Albumin-BindingMaytansinoids 30 and 31 in the Human PDX Squamous Cell Lung Cancer ModelLXFE 937 (Large Tumors)

The evaluation of maytansine and the albumin-binding compounds 30 and 31in the squamous cell lung cancer LXFE 937 model was carried outaccording to the general procedure for a patient-derived xenograftmodel. Treatment was initiated after tumors reaching an average size of270 mm³. FIG. 7 shows tumor growth curves of the control group,maytansine group, and the groups treated with compounds 30 and 31. FIG.8 shows curves of body weight change in the control group, maytansinegroup, and the groups treated with compounds 30 and 31.

Example 16 Evaluation of Maytansine and the Albumin-BindingMaytansinoids 30 and 31 in the Human PDX Lung Adenocarcinoma Model LXFA737

The evaluation of maytansine and the albumin-binding compounds 30 and 31in the lung adenocarcinoma LXFA 737 model was carried out according tothe general procedure for a patient-derived xenograft model. Treatmentwas initiated after tumors reaching an average size of 311 mm³. FIG. 9shows tumor growth curves of the control group, maytansine group, andthe groups treated with compounds 30 and 31. FIG. 10 shows curves ofbody weight change in the control group, maytansine group, and thegroups treated with compounds 30 and 31.

Example 17 Evaluation of Maytansine and the Albumin-BindingMaytansinoids 32, 30 and 31 in the Human Xenograft Breast CarcinomaModel MDA-MB-231

The evaluation of maytansine and the albumin-binding compounds 32, 30and 31 in the MDA-MB 231 breast carcinoma model was carried outaccording to the general procedure for a cancer cell-line-derivedxenograft model. Treatment was initiated after tumors reaching anaverage size of 80 mm³. FIG. 11 shows tumor growth curves of the controlgroup, maytansine group, and the groups treated with compounds 32, 30,and 31. FIG. 12 shows curves of body weight change in the control group,maytansine group, and the groups treated with compounds 32, 30, and 31.

Example 18 Evaluation of Maytansine and the Albumin-BindingMaytansinoids 30 and 31 in a Human Xenograft Ovarian Cancer Model A2780

The evaluation of maytansine and the albumin-binding compounds 30 and 31in ovarian cancer A2780 model was carried out according to the generalprocedure for a cancer cell-line-derived xenograft model. Treatment wasinitiated after tumors reaching an average size of 380 mm³. FIG. 13shows tumor growth curves of the control group, maytansine group, andthe groups treated with compounds 30 and 31. FIG. 14 shows curves ofbody weight change in the control group, maytansine group, and thegroups treated with compounds 30 and 31.

Example 19 Evaluation of Maytansine and the Albumin-BindingMaytansinoids 30 and 31 in the Human Xenograft Breast Carcinoma ModelMDA-MB 468

The evaluation of maytansine and the albumin-binding compounds 30 and 31in breast carcinoma MDA-MB 468 model was carried out according to thegeneral procedure for a cancer cell-line-derived xenograft model.Treatment was initiated after tumors reaching an average size of 94 mm³.FIG. 15 shows tumor growth curves of the control group, maytansinegroup, and the groups treated with compounds 30 and 31. FIG. 16 showscurves of body weight change in the control group, maytansine group, andthe groups treated with compounds 30 and 31.

1. A compound having the structure of Formula (I):

or a pharmaceutically acceptable salt, hydrate, solvate, or isomerthereof, wherein: R¹ is selected from —H and C₁-C₄ alkyl; Spacer isselected from:

V is absent or selected from —CH₂—, —O— and —NR³—, wherein R³ is —H orC₁-C₄ alkyl; each R² is independently selected from —H, halogen (e.g.,—F, —Cl, —Br or —I) and C₁-C₄ alkyl or two R²s taken together form aC₃-C₆, cycloalkyl; n is 0-3; X is absent or selected from —CH₂—, —O—,—S—, —Se—, and —NR⁴—, wherein R⁴ is —H or C₁-C₄ alkyl; Y is selectedfrom ═CH— and ═N—; Z¹, Z², Z³ and Z⁴ are each independently selectedfrom —H, halogen (e.g., —F, —Cl, —Br or —I), —CF₃, —OCH₃, —CN, —NO₂,C₁-C₄ alkyl and C₂-C₄ alkoxy; AA is an amino acid selected from glycine,D or L proline, sarcosine, N-ethyl-glycine, D or L alanine, D or LN-methylalanine, β-alanine, N-methyl-β-alanine, α-aminoisobutyric acid,and N-methyl-α-aminoisobutyric acid; R′ is selected from O and

Y′ is absent or selected from an optionally substituted C₁-C₆ alkyl,—NH—C(O)—, and —C(O)—NH—; or Y′ is selected from the group consistingof:

wherein n=0-6; R^(1′) is absent or selected from the group consistingof:

wherein M¹ is a pharmaceutically acceptable counter ion (e.g., H⁺, Na⁺,K⁺, Ca²⁺, Mg²⁺, NR₄ ⁺, and NHR₃ ⁺; wherein R is H or C₁-C₄ alkyl);R^(2′) is optionally substituted C₁-C₁₈ alkyl wherein optionally up tosix carbon atoms in said C₁-C₁₈ alkyl are each independently replacedwith —OCH₂CH₂—; Z^(1′), Z^(2′), Z^(3′) and Z^(4′) are each independentlyselected from —H, halogen (e.g., —F, —Cl, —Br or —I), —CF₃, —OCH₃, —CN,—NO₂, —SO₃M², and C₁-C₄ alkyl wherein M² is a pharmaceuticallyacceptable counter ion (e.g., H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, NR₄ ⁺, and NHR₃⁺; wherein R is H or C₁-C₄ alkyl); TBG is a thiol-binding group selectedfrom an optionally substituted maleimide group, an optionallysubstituted haloacetamide group, an optionally substituted haloacetategroup, an optionally substituted pyridylthio group, an optionallysubstituted isothiocyanate group, an optionally substitutedvinylcarbonyl group, an optionally substituted aziridine group, anoptionally substituted disulfide group, an optionally substitutedacetylene group, and an optionally substituted N-hydroxysuccininideester group; wherein said TBG is optionally bound to a thiol-bearingmacromolecular carrier or thiol-bearing tumor-specific carrier. 2.-36.(canceled)
 37. The compound of claim 1, wherein said compound isselected from the group consisting of:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 38.The compound of claim 37, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 39.The compound of claim 37, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 40.The compound of claim 37, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 41.The compound of claim 37, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 42.The compound of claim 37, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 43.The compound of claim 37, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 44.The compound of claim 37, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 45.The compound of claim 37, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 46.The compound of claim 37, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 47.The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 48.The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 49.The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 50.The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 51.The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 52.The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 53.The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 54.The compound of claim 1, wherein the compound is

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 55.The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 56.The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 57.The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof. 58.The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt, hydrate, or isomer thereof.
 59. Apharmaceutical composition comprising the compound, of claim 37, orpharmaceutically acceptable salt, hydrate, or isomer thereof, and apharmaceutically acceptable carrier.
 60. A pharmaceutical compositioncomprising the compound, of any one of claim claim 47-58, orpharmaceutically acceptable salt, hydrate, or isomer thereof, and apharmaceutically acceptable carrier.
 61. A method for treating cancer,comprising administering to a patient in need thereof a therapeuticallyeffective amount of the pharmaceutical composition according to claim59, wherein the cancer is selected from the group consisting ofadenocarcinoma, uveal melanoma, acute leukemia, acoustic neuroma,ampullary carcinoma, anal carcinoma, astrocytoma, basalioma, pancreaticcancer, connective tissue tumor, bladder cancer, bronchial carcinoma,non-small cell bronchial carcinoma, breast cancer, Burkitt's lymphoma,corpus carcinoma, CUP syndrome, colon cancer, cancer of the smallintestine, ovarian cancer, endometrial carcinoma, gallbladder cancer,gallbladder carcinomas, uterine cancer, cervical cancer, neck, nose andear tumors, hematological neoplasia, hairy cell leukemia, urethralcancer, skin cancer, gliomas, testicular cancer, Kaposi's sarcoma,laryngeal cancer, bone cancer, colorectal carcinoma, head/neck tumors,colon carcinoma, craniopharyngeoma, liver cancer, leukemia, lung cancer,non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma,stomach cancer, colon cancer, medulloblastoma, melanoma, meningioma,kidney cancer, renal cell carcinomas, oligodendroglioma, esophagealcarcinoma, osteolytic carcinomas and osteoplastic carcinomas,osteosarcoma, ovarian carcinoma, pancreatic carcinoma, penile cancer,prostate cancer, tongue cancer, ovary carcinoma, and lymph gland cancer.62. A method for treating cancer, comprising administering to a patientin need thereof a therapeutically effective amount of the pharmaceuticalcomposition according to claim 60, wherein the cancer is selected fromthe group consisting of adenocarcinoma, uveal melanoma, acute leukemia,acoustic neuroma, ampullary carcinoma, anal carcinoma, astrocytoma,basalioma, pancreatic cancer, connective tissue tumor, bladder cancer,bronchial carcinoma, non-small cell bronchial carcinoma, breast cancer,Burkitt's lymphoma, corpus carcinoma, CUP syndrome, colon cancer, cancerof the small intestine, ovarian cancer, endometrial carcinoma,gallbladder cancer, gallbladder carcinomas, uterine cancer, cervicalcancer, neck, nose and ear tumors, hematological neoplasia, hairy cellleukemia, urethral cancer, skin cancer, gliomas, testicular cancer,Kaposi's sarcoma, laryngeal cancer, bone cancer, colorectal carcinoma,head/neck tumors, colon carcinoma, craniopharyngeoma, liver cancer,leukemia, lung cancer, non-small cell lung cancer, Hodgkin's lymphoma,non-Hodgkin's lymphoma, stomach cancer, colon cancer, medulloblastoma,melanoma, meningioma, kidney cancer, renal cell carcinomas,oligodendroglioma, esophageal carcinoma, osteolytic carcinomas andosteoplastic carcinomas, osteosarcoma, ovarian carcinoma, pancreaticcarcinoma, penile cancer, prostate cancer, tongue cancer, ovarycarcinoma, and lymph gland cancer.